CN1942589A - Modified human growth hormone polypeptides and uses thereof - Google Patents

Abstract

The present invention provides modified human growth hormone polypeptides and uses thereof.

Description

Modified human interferon polypeptides and its purposes

The present invention advocates the U.S. Provisional Patent Application case the 60/541st of application on February 2nd, 2004, the U.S. Provisional Patent Application case the 60/581st of application on June 18th, No. 528 1, the U.S. Provisional Patent Application case the 60/581st of application on June 18th, No. 314 1, the U.S. Provisional Patent Application case the 60/580th of application on June 18th, No. 175 1, the application of No. 885 and on December 22nd, 2004 be entitled as 60/638, the right of priority of 616 U.S. Provisional Patent Application case, the mode that the specification sheets of described application case is quoted in full is incorporated herein.

Technical field

The present invention relates to growth hormone polypeptides through at least a non-naturally encoded amino acids modification.

Background technology

Tethelin (GH) supergene family (Bazan, F.Immunology Today 11:350-354 (1991); Mott, H.R. and Campbell, I.D.Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N. (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS) represent one group of albumen with similar structures feature.Each member of this protein family comprises four-helix bundle, and it is the universal architecture of person shown in Fig. 1.Though more members of described family are still unidentified, some members of described family comprise following each thing: tethelin, prolactin, galactagogin, erythropoietin (EPO), thrombopoietin (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukaemia inhibitory factor, interferon-alpha, interferon-, IFN-, omega interferon, the τ Interferon, rabbit, the ε Interferon, rabbit, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage group stimulating factor (GM-CSF), scavenger cell group stimulating factor (M-CSF) and heart nutrient substance-1 (CT-1) (" GH supergene family ").The member of GH supergene family has similar secondary and tertiary structure, though it has limited amino acid or consensus dna sequence usually.The feasible newcomer who is easy to discern gene family of total constitutional features.The universal architecture of family member hGH, EPO, IFN α-2 and G-CSF is showed in respectively among Fig. 2,3,4 and 5.

A member of GH supergene family is human growth hormone (hGH).Many regulation and control that the human growth hormone participates in the normal human subject growth and grows.The molecular weight of about 22kDa formed and had by the strand pituitrin of this natural generation by 191 amino-acid residues.HGH represents multiple biological effect, especially comprises linear growth (physique generation), lactogenesis, activated macrophage and para-insulin and diabetogenic effect (Chawla, people such as R., Ann.Rev.Med.34:519-547 (1983); Isaksson, people such as O., Ann.Rev.Physiol., 47:483-499 (1985); Hughes, J. and Friesen, H., Ann.Rev.Physiol, 47:469-482 (1985)).

The structure of hGH is well-known (Goeddel, people such as D., Nature 281:544-548 (1979)), and the three-dimensional structure of hGH is resolved (de Vos, people such as A., Science 255:306-312 (1992)) by X-ray crystallography.Albumen has compact ball-like structure, and it comprises four both sexes α helical bundles, and from the initial A-D that is called as of N-terminal, it is by engagement of loops.HGH also contains four cysteine residues, and it participates in two intramolecular disulfide bond: C53 and C165 pairing and C182 and C189 pairing.Hormone and without glycosylation and in intestinal bacteria, expressed (Chang, people such as C, Gene 55:189-196 (1987)) with secreted form.

Discerned the mutant of the multiple natural generation of hGH.These mutant comprise hGH-V (Seeberg, DNA 1:239 (1982); United States Patent (USP) the 4th, 446,235,4,670,393 and 4,665, No. 180, it is incorporated herein by reference) and contain 20-kDa hGH (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of the residue 32-46 disappearance of hGH; Lewis, people such as U., J.Biol Chem., 253:2679-2687 (1978)).In addition, reported multiple hGH varient, it is to produce (Baumann, G., Endocrine Reviews 12:424 (1991)) by transcribing back, translation back, secretion property, metabolism processed-type and other physiological process.

The biological effect of hGH is the interaction that is derived from itself and specific cell acceptor.Hormone is the member who comprises the homologous protein family of galactagogin and prolactin.Yet, hGH is uncommon in the family member, because its represent species specificity widely and with the former (Leung of body through the clone, D. wait the people, Nature 330:537-543 (1987)) acceptor or prolactin (Boutin, J. waiting the people, Cell 53:69-77 (1988)) acceptor combines.Based on structure and Biochemical Research, lactagogue and the body originality function collection of illustrative plates (Cunningham, B. and Wells, J., Proc.Natl.Acad.Sci.88:3407 (1991)) in conjunction with the territory has been proposed.The hGH acceptor is a member of hematopoiesis/cytohormone/growth factor receptors family, described family comprises several other growth factor receptorses, such as interleukin (IL)-3 ,-4 and-6 acceptors, granular leukocyte macrophage group stimulating factor (GM-CSF) acceptor, erythropoietin (EPO) acceptor and G-CSF acceptor.Referring to Bazan, Proc.Natl.Acad.Sci USA 87:6934-6938 (1990).The member of cytohormone receptor family is contained four conservative property cysteine residues and just in time is positioned at the tryptophane-Serine-X-tryptophane-Serine primitive of striding the outside, film district.Think that the conservative property sequence is involved in the protein-protein interaction.For example referring to people such as Chiba, Biochim.Biophys.Res.Comm.184:485-490 (1992).Interaction between the extracellular domain of hGH and its acceptor (hGHbp) is to be most one of hormone-receptor interaction that the people understood.High-res X-ray crystallography data (Cunningham, people such as B., Science, 254:821-825 (1991)) have showed that hGH has two receptor binding sites and uses site unique on the molecule successively in conjunction with two acceptor molecules.Described two receptor binding sites are called as site I and site II.Site I comprises the C-terminal of spiral D and part spiral A and A-B ring, and the amino that site II is contained spiral A not petiolarea and part spiral C.GH takes place with combining successively of its acceptor, at first combination of site I.Site II meshes the 2nd GH acceptor then, causes the receptor dimerization effect and causes activation to the intracellular signaling pathway of the cell response of hormone.The hGH mutain of wherein G120R replace having been introduced among the II of site can be in conjunction with single hGH acceptor, but can not two kinds of acceptors of dimerization.Mutain serves as external hGH antagonist, infer its be by occupy acceptor site simultaneously can active cells in signal path (Fuh, people such as G., Science 256:1677-1680 (1992)).

Reorganization hGH is used for the treatment of multiple indication as therapeutical agent and approved.The hGH shortage causes nanism, and for example it comes into operation hormone by external source and successfully treats more than 10 years.Except that hGH lacked, hGH also was approved for the emaciation among treatment renal failure (among the children), Turner's synodrome and the AIDS patient.Recently, to be used for the treatment of non-GH dependency of short and small stature for food and FAD (FDA) approved hGH.HGH is current also to be studied to be used for the treatment of aging, the elderly's weakness, short bowel syndrome and congestive heart failure.

The hGH that recombinates is current to be that currently marketed five kinds of main productss are: Humatrope as injectable product sale every day^TM(Eli Lilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono).Yet, has the transformation period in the short body for a remarkable difficult problem of using tethelin as therapeutical agent as albumen, therefore and it must come into operation to reach maximum efficiency (people such as MacGillivray, J.Clin.Endocrinol.Metab.81:1806-1809 (1996) by subcutaneous injection every day.Significantly make great efforts be concentrate on that dispensing is easier for patients by reducing production costs, making, improvement effect and security overview and produce and will provide other characteristic of competitive edge to improve coming into operation of hGH agonist and antagonist.For example, Genentech and Alkermes had before sold Nutropin Depot^TM, it is that the hGH that is used for the paediatrics growth hormone deficiency stores composite.Though described storage thing allows more low-frequency dispensing (every 2-3 week once, rather than once a day), it is also relevant with side effect improperly, such as the biological utilisation and the injection site place pain that reduce, and withdraws from market in 2004.Another product P egvisomant^TM(Pfizer) also ratify by FDA recently.Pegvisomant^TMBe the genetically engineered analogue of hGH, it is as specifying the high selectivity growth hormone receptor antagonist be used for the treatment of acromegaly (people such as van der Lely, The Lancet 358:1754-1759 (2001).Though Pegvisomant^TMIn several amino acid side chain residues through polyoxyethylene glycol (PEG) polymer-derived, but described product still once a day the dispensing, show that medicinal properties is not the best.Except that Pegylation and storage composite, comprise that the suction of hGH and other dosing way of oral dosage form are in clinical preceding initial stage and clinical development stage, and do not have dosing way to ratify as yet by FDA.Therefore, it is active and long serum half-life is provided and therefore provides better hGH treatment level and the polypeptide of the therapeutical agent transformation period of increase to represent tethelin.

Poly-(ethylene glycol) (being abbreviated as PEG) of covalently bound hydrophilic polymer is a kind of method that increases water-soluble and biological utilisation, increases serum half-life, increases the therapeutical agent transformation period, regulates immunogenicity, regulates biological activity or prolong the cycling time of many bioactive moleculess (comprising albumen, peptide and especially hydrophobic molecule).PEG has been widely used in the medicine, be used for the artificial graft goes up and is used for wherein biocompatibility, toxicity and lack other that lack tool importance with immunogenicity and use.The characteristic of being wanted by maximization PEG, the total molecular weight of the PEG polymkeric substance that connects with bioactive molecules and hydration status enough height be connected relevant favorable characteristics to give usually with the PEG polymkeric substance, such as the water-soluble and circulating half-life that increases, and can influence the biological activity of parent molecule sharply.

The PEG derivative often is connected with bioactive molecules via the chemical functional group of reactivity, and described functional group is such as being Methionin, halfcystine and histidine residues, N-terminal and carbohydrate part.Albumen and other molecule have a limited number of reactive site that polymkeric substance connects that can be used for usually.Usually, be suitable for connecting the site of modifying most and in receptors bind, play an important role via polymkeric substance, and essential by the reservation of molecular biological activity.Therefore, even being connected the biological activity that causes usually through polymer-modified molecule with the indifference of the above reactive site of bioactive molecules, polymer chain significantly reduces complete loss.People such as R.Clark, (1996),J.Biol.Chem.,271:21969-21977.For having, formation is enough to give the joiner that target molecule is wanted the polymericular weight of advantage, prior art approach has comprised usually numerous polymeric arms has been connected in described molecule at random, reduces the risk of loss fully even increase the biological activity of parent molecule thus.

The reactive site that is formed for the PEG derivative is connected in proteic locus is arranged by protein structure.The albumen that comprises enzyme is by having universal architecture H₂The various alpha amino acid sequences of N--CHR--COOH are formed.The amino part of a kind of amino acid whose α (H₂N--) join contiguous amino acid whose carboxy moiety to (--COOH) to form amido linkage, it can be expressed as--(NH--CHR--CO)_n--wherein subscript " n " can equal hundreds of or several thousand.Fragment by the R representative can contain the reactive site that is useful on protein biological activity and is used to connect the PEG derivative.

For example, under the situation of amino acid lysine, in ε position and alpha position, exist--NH₂Part.ε under the alkaline pH condition--NH₂Do not react.Proteic PEG derive in the field most of technology at exploitation PEG derivative to be used for being connected in the ε of the existing lysine residue of albumen--NH₂Part." Polyethylene Glycoland Derivatives for Advanced PEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages or leaves.Yet these PEG derivatives all have common limitation, and promptly it can not selectivity be installed on the protein surface in existing numerous usually lysine residue.Lysine residue to the situation that for example is present in the protein-active tool importance in the enzyme active sites under, perhaps regulating under the situation about working aspect the interaction of albumen and other biomolecules (under the situation at receptor binding site) at lysine residue, it can be remarkable restriction.

The complexcase second largest and of equal importance of existing albumen Pegylation method are that the PEG derivative can experience improper side reaction with the residue except that want person.Histidine contains reactive imino-part (structurally being expressed as--N (H)--), but many and ε--NH₂The chemical reactivity material of reaction also can react with--N (H)--.Similarly, the side chain of amino acid cysteine has free sulfhydryl groups, and it structurally is expressed as-SH.In some cases, at the ε-NH of Methionin₂Aligned PEG derivative also reacts with halfcystine, Histidine or other residue on the group.So can produce complex heterogeneous mixture through PEG deutero-bioactive molecules, and might destroy at the activity of bioactive molecules.Need the single site of exploitation permission in albumen to introduce chemical functional group's PEG derivative, it makes that then one or more PEG polymkeric substance can be through clearly definition and predictable specificity site selectivity are coupled to bioactive molecules on protein surface.

Except that lysine residue, in affiliated field, split the activated PEG reagent input remarkable effort of hairpin to other amino acid chain (comprising halfcystine, Histidine and N-terminal).For example referring to United States Patent (USP) the 6th, 610, No. 281 (it is incorporated herein by reference) and " Polyethylene Glycol and Derivatives for AdvancedPEGylation ", Nektar Molecular Engineering Catalog, 2003, the 1-17 pages or leaves.Use known other technology in rite-directed mutagenesis forming method and the affiliated field, cysteine residues site selectivity ground can be introduced in the protein structure, and the gained free sulfhydryl groups partly can with the PEG derivatives reaction with thiol-reactive functional group.Yet this method is very complicated, can make the proteic expression of gained, folding and stability become complicated because introduce free sulfhydryl groups.Therefore, need have chemical functional group is introduced mode in the bioactive molecules, described bioactive molecules can make one or more PEG polymkeric substance and the coupling of albumen selectivity, simultaneously with albumen in the common sulfydryl of finding and other chemical functional group compatible (promptly not participating in improper side reaction) with it.

Can see from the sampling in affiliated field, be used to be connected on albumen side chain, especially the Methionin amino acid side chain through exploitation--NH₂On part and the cysteine side chain-SH many described derivative partly is verified to have problems on it synthesizes and uses.Some derivatives and albumen form unsettled bonding, its in aqueous environment (such as in blood flow) stand hydrolysis and therefore decompose, degraded or in the others instability.Some derivatives form more stable bonding, but still stand hydrolysis before forming bonding, and its reactive group that means on the PEG derivative can be through inactivation before albumen connects.Some derivatives have a little toxicity and therefore are not suitable for using in the body.Some derivatives reactions are inapplicable too slowly and actually.Some derivatives cause the protein-active loss by being connected in the site of being responsible for protein-active.Some derivatives do not have specificity, the reproducibility that it also can cause institute's loss of activity of wanting and lack the result at it in the site that connects.For overcome with the poly-relevant difficult problem of (ethylene glycol) part modified protein, developed that stable (for example United States Patent (USP) the 6th, 602, No. 498, it is incorporated herein by reference) or with molecule and lip-deep thiol moiety selective reaction (for example United States Patent (USP) the 6th, 610, No. 281, it is incorporated herein by reference) the PEG derivative.In affiliated field obviously need up to be waken up with selective reaction with form stablize chemical bond before in physiological environment always for chemically inert PEG derivative.

Recently, reported complete novel technology in the albumen science, it gets a good chance of overcoming the relevant limitation of many and proteic site-specific sex modification.Specific, to prokaryotic organism intestinal bacteria (E.coli) (people such as L.Wang for example, (2001),Science292:498-500) and eukaryote yeast saccharomyces cerevisiae (S.cerevisiae) (people such as J.Chin for example,Science301:964-7 (2003)) add novel components in the protein biology combination mechanism, it makes it possible to and will incorporate in the albumen in the non-genomic amino acids coding body.Use this method, multiple novel amino amino acid, ketone group amino acid and the glycosylation amino acid of photoaffinity labeling and photoisomerization (but comprise) that will have novel chemistry, physics or biological nature reaches high frequency high fidelity ground effectively to be incorporated in intestinal bacteria and in the yeast in the albumen in response to amber codon TAG.For example referring to people such as J.W.Chin, (2002),Journal of the American Chemical Society124:9026-9027; J.W.Chin ， ﹠amp; P.G.Schultz, (2002),ChemBio Chem11:1135-1137; People such as J.W.Chin, (2002),PNAS United States of America99:11020-11024; With L.Wang ， ﹠amp; P.G.Schultz, (2002),Chem.Comm.,1-10.These researchs are verified might selectivity and introduce chemical functional group routinely, such as ketone group, alkynyl and nitrine part, it finds in albumen, be unreactiveness and can be used for effectively and optionally reacting to form stable covalent bonding for all functional groups that found in the amino acid of 20 kinds of common genes encodings.

Incorporate the non-genomic amino acids coding into chemical functional group that ability in the albumen allow to be introduced the functional group's that natural generation can be provided valuable surrogate, such as the ε-NH of Methionin₂, sulfydryl-SH of halfcystine, the imino-of Histidine etc.Known some chemical functional group is inertia for the functional group who is found in the amino acid of 20 kinds of common genes encodings, but significantly and effectively reacts to form stable bonding.For example, known azido-and acetylene are experiencing Huisgen [3+2] cycloaddition reaction under aqueous conditions in the presence of the copper of catalytic amount in affiliated field.For example referring to people such as Tornoe, (2002)Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002)Angew.Chem.Int.Ed.41:2596-2599.For example,, can incorporate into and be unreactiveness, but also react smoothly and effectively to form the functional group of cycloaddition product with acetylene moiety for the amine of in albumen, being found, sulfydryl, carboxylic acid, hydroxyl by nitrine is partly introduced in the protein structure.Importantly, under the situation that does not have acetylene moiety, azido-is in the presence of other albumen side chain and still be unreactiveness and anergy under the physiological condition.

The present invention especially solves the problem relevant with generation with the activity of GH polypeptide, also solves the generation of the hGH polypeptide with improvement biology or pharmacological characteristics (such as the therapeutical agent transformation period of improvement).

Summary of the invention

The invention provides GH supergene family member, it comprises the hGH polypeptide that comprises one or more non-naturally encoded amino acids.

In certain embodiments, the hGH polypeptide comprises one or more posttranslational modifications.In certain embodiments, the hGH polypeptide is connected with connexon, polymkeric substance or bioactive molecules.In certain embodiments, the hGH polypeptide is connected with double functional copolymer, difunctionality connexon or at least a extra hGH polypeptide.

In certain embodiments, non-naturally encoded amino acids is connected with water-soluble polymers.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, poly-(ethylene glycol) molecule is the double functional copolymer.In certain embodiments, the double functional copolymer is connected with second polypeptide.Second polypeptide is the hGH polypeptide in certain embodiments.

In certain embodiments, the hGH polypeptide comprises at least two kinds of amino acid that are connected with the water-soluble polymers that comprises poly-(ethylene glycol) part.In certain embodiments, at least a amino acid is non-naturally encoded amino acids.

The zone of hGH can be described as follows, and wherein specifies the amino acid position among the hGH in the row of centre:

Spiral A spiral B spiral C spiral D

[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184-191]

N-terminal A-B ring B-C ring C-D ring C-terminal.

In certain embodiments, incorporate one or more non-naturally encoded amino acids in following any position: the corresponding amino acid whose 1-5 (N-terminal) of SEQ ID NO:2 or SEQ ID NO:1 or 3 with in one or more of lower area corresponding to the secondary structure among the hGH, 6-33 (A spiral), 34-74 (the zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), 97-105 (the zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-terminal).In other embodiments, replace non-naturally encoded amino acids in the position of the group that the corresponding amino acid whose residue 1-5,32-46,97-105,132-149 and the 184-191 that are selected from by hGH SEQ ID NO:2 or SEQ ID NO:1 or 3 form.In certain embodiments, in hGH to incorporate one or more non-naturally encoded amino acids in one or more of upper/lower positions into: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

In certain embodiments, to replace one or more non-naturally encoded amino acids in one or more of upper/lower positions: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

In certain embodiments, to replace one or more non-naturally encoded amino acids in one or more of upper/lower positions: 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 or 187 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

In certain embodiments, to replace one or more non-naturally encoded amino acids in one or more of upper/lower positions: 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

In certain embodiments, to replace one or more non-naturally encoded amino acids in one or more of upper/lower positions: 30,74 or 103 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, to replace one or more non-naturally encoded amino acids in one or more of upper/lower positions: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

In certain embodiments, non-naturally encoded amino acids in one or more of these positions is connected in water-soluble polymers, includes, but is not limited to upper/lower positions: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the non-naturally encoded amino acids in one or more of these positions is connected in water-soluble polymers: 30,35,74,92,103,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ IDNO:1 or 3).In certain embodiments, the non-naturally encoded amino acids in one or more of these positions is connected in water-soluble polymers: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

Human GH antagonist includes, but is not limited to have 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 and replaces or 1 (being N-terminal) has and add or the antagonist of its any combination (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3 or any other GH sequence) in the position.

In certain embodiments, the hGH polypeptide comprises replacement, interpolation or the disappearance of adjusting hGH polypeptide to the avidity of hGH polypeptide receptor.In certain embodiments, the hGH polypeptide comprises replacement, interpolation or the disappearance that increases hGH polypeptide stability.In certain embodiments, the hGH polypeptide comprises in hGH SEQ ID NO:2 and is selected from by F10A, F10H, F10I; M14W, M14Q, M14G; H18D, H21N, G120A; R167N, D171S, E174S; The aminoacid replacement of the group that F176Y, I179T or its any combination are formed.In certain embodiments ,+the hGH polypeptide comprises immunogenic replacement, interpolation or the disappearance of regulating the hGH polypeptide.In certain embodiments, the hGH polypeptide comprises serum half-life or the replacement of cycling time, interpolation or the disappearance of regulating the hGH polypeptide.

In certain embodiments, the hGH polypeptide comprises water miscible replacement, interpolation or the disappearance that increases the hGH polypeptide.In certain embodiments, the hGH polypeptide comprises replacement, interpolation or the disappearance of the solubleness that is increased in the hGH polypeptide that produces in the host cell.In certain embodiments, the hGH polypeptide comprises increases in the host cell or replacement, interpolation or the disappearance of external synthetic hGH polypeptide expression.In certain embodiments, the hGH polypeptide comprises aminoacid replacement G120A.Comprise the active also reservation of hGH polypeptide reservation agonist of this replacement or the expression level in the improvement host cell.In certain embodiments, the hGH polypeptide comprises replacement, interpolation or the disappearance of the protease resistant that increases the hGH polypeptide.

In certain embodiments, the aminoacid replacement in the hGH polypeptide can natural generation or non-natural produce amino acid and carry out, as long as at least one replacement is with non-naturally encoded amino acids.

In certain embodiments, non-naturally encoded amino acids comprises carbonyl, aminooxy, diazanyl, hydrazide group, amino urea groups, azido-or alkynyl.

In certain embodiments, non-naturally encoded amino acids comprises carbonyl.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R₃For H, amino acid, polypeptide or N-terminal are modified base, and R₄For H, amino acid, polypeptide or C-terminal are modified base.

In certain embodiments, non-naturally encoded amino acids comprises aminooxy.In certain embodiments, non-naturally encoded amino acids comprises hydrazide group.In certain embodiments, non-naturally encoded amino acids comprises diazanyl.In certain embodiments, the non-naturally encoded amino acids residue comprises amino urea groups.

In certain embodiments, the non-naturally encoded amino acids residue comprises azido-.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.

In certain embodiments, non-naturally encoded amino acids comprises alkynyl.In certain embodiments, non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl or be substituted aryl; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.

In certain embodiments, polypeptide is hGH polypeptide agonist, part agonist, antagonist, partial antagonist or reverse agonist.In certain embodiments, hGH polypeptide agonist, part agonist, antagonist, partial antagonist or reverse agonist comprise the non-naturally encoded amino acids that is connected in water-soluble polymers.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, hGH polypeptide agonist, part agonist, antagonist, partial antagonist or reverse agonist comprise non-naturally encoded amino acids and one or more posttranslational modifications, connexon, polymkeric substance or bioactive molecules.In certain embodiments, the non-naturally encoded amino acids that is connected in water-soluble polymers is present in the II zone, site (the albumen zone of containing amino terminal region and the part spiral C of AC helical bundle face, spiral A) of hGH polypeptide.In certain embodiments, the hGH polypeptide that comprises the non-naturally encoded amino acids that is connected in water-soluble polymers is attached to the dimerisation that the 2nd hGH polypeptide receptor molecule stops the hGH polypeptide receptor by stoping the hGH polypeptide antagonist.In certain embodiments, the aminoacid replacement G120 of usefulness except that glycine in SEQ ID NO:2 (hGH).In certain embodiments, in SEQ ID NO:2, replace G120 with arginine.In certain embodiments, in SEQ ID NO:2, replace G120 with non-naturally encoded amino acids.

The present invention also provide be included in the polynucleotide that hybridize to SEQ ID NO:21 or 22 under the rigorous condition through isolating nucleic acid, wherein said polynucleotide comprise at least one and select codon.In certain embodiments, selecting codon is to be selected from by amber codon, ocher codon, opal codon, unique password, rare codon and the molecular group of four base passwords.

The present invention also provides the method for making the hGH polypeptide that is connected in water-soluble polymers.In certain embodiments, described method comprise with water-soluble polymers contact comprise non-naturally encoded amino acids through separating the hGH polypeptide, described water-soluble polymers comprises the part with the non-naturally encoded amino acids reaction.In certain embodiments, incorporate in the hGH polypeptide non-naturally encoded amino acids to can not be in addition with 20 kinds of common amino acids in the water-soluble polymers of any reaction have reactivity.In certain embodiments, incorporate in the hGH polypeptide non-naturally encoded amino acids to can not be in addition with 20 kinds of common amino acids in connexon, polymkeric substance or the bioactive molecules of any reaction have reactivity.

In certain embodiments, the hGH polypeptide that is connected in water-soluble polymers is made by comprising the hGH polypeptide that contains carbonylamino acid and poly-(ethylene glycol) molecular reaction that comprises aminooxy, diazanyl, hydrazide group or amino urea groups.In certain embodiments, aminooxy, diazanyl, hydrazide group or amino urea groups are connected in poly-(ethylene glycol) molecule via amido linkage.

In certain embodiments, the hGH polypeptide that is connected in water-soluble polymers is made by the polypeptide reaction of poly-(ethylene glycol) molecule that will comprise carbonyl and the non-naturally encoded amino acids that comprises aminooxy, diazanyl, hydrazide group or amino urea groups.

In certain embodiments, the hGH polypeptide that is connected in water-soluble polymers is made by comprising the hGH polypeptide that contains alkynyl amino acid and poly-(ethylene glycol) molecular reaction that comprises the nitrine part.In certain embodiments, azido-or alkynyl are connected in poly-(ethylene glycol) molecule via amido linkage.

In certain embodiments, the hGH polypeptide that is connected in water-soluble polymers is to contain the amino acid whose hGH polypeptide of azido-and made with poly-(ethylene glycol) molecular reaction that comprises alkynyl moiety by comprising.In certain embodiments, azido-or alkynyl are connected in poly-(ethylene glycol) molecule via amido linkage.

In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between about 0.1kDa and about 100kDa.In certain embodiments, poly-(ethylene glycol) molecule has the molecular weight between 0.1kDa and 50kDa.

In certain embodiments, poly-(ethylene glycol) molecule is a branched polymers.In certain embodiments, each side chain of poly-(ethylene glycol) branched polymers has between between 1kDa and the 100kDa or the molecular weight between 1kDa and 50kDa.

In certain embodiments, the water-soluble polymers that is connected in the hGH polypeptide comprises polyalkylene glycol moiety.In certain embodiments, the non-naturally encoded amino acids residue of incorporating in the hGH polypeptide comprises carbonyl, aminooxy, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl.In certain embodiments, incorporate that non-naturally encoded amino acids residue in the hGH polypeptide comprises carbonyl moiety and water-soluble polymers comprises aminooxy, hydrazides, hydrazine or Urea,amino-part into.In certain embodiments, incorporate that non-naturally encoded amino acids residue in the hGH polypeptide comprises alkynyl moiety and water-soluble polymers comprises the nitrine part into.In certain embodiments, incorporate that non-naturally encoded amino acids residue in the hGH polypeptide comprises the nitrine part and water-soluble polymers comprises alkynyl moiety into.

The present invention also provides composition, and it comprises the hGH polypeptide of non-naturally encoded amino acids and pharmaceutically acceptable supporting agent.In certain embodiments, non-naturally encoded amino acids is connected with water-soluble polymers.

The present invention also provides cell, and it comprises the polynucleotide of the coding hGH polypeptide of selecting codon.In certain embodiments, described cell comprises and is used for non-naturally encoded amino acids is replaced quadrature RNA synthetic enzyme and/or quadrature tRNA into the hGH polypeptide.

The present invention also provides the method for making the hGH polypeptide that comprises non-naturally encoded amino acids.In certain embodiments, described method is included in the cell of cultivating the polynucleotide, quadrature RNA synthetic enzyme and/or the quadrature tRNA that comprise coding hGH polypeptide under the condition that allows the hGH expression of polypeptides and purifying hGH polypeptide from cell and/or substratum.

The present invention also provides the method for the therapeutical agent transformation period, serum half-life or the cycling time that increase the hGH polypeptide.The present invention also provides the immunogenic method of regulating the hGH polypeptide.In certain embodiments, described method comprises with non-naturally encoded amino acids and replaces any or more than one amino acid in the hGH polypeptide of natural generation and/or the hGH polypeptide chain is connected to connexon, polymkeric substance, water-soluble polymers or bioactive molecules.

The present invention also provides the method that needs the patient of described treatment with the hGH molecular therapy of the present invention of significant quantity.In certain embodiments, described method comprise to the patient come into operation the treatment significant quantity medical composition, described medical composition comprises the hGH polypeptide of non-naturally encoded amino acids and pharmaceutically acceptable supporting agent.In certain embodiments, non-naturally encoded amino acids is connected with water-soluble polymers.

The present invention also provide comprise SEQ ID NO:1,2,3 or any other GH peptide sequence shown in the hGH polypeptide of sequence, its exception is replaced by non-naturally encoded amino acids at least a amino acid.In certain embodiments, non-naturally encoded amino acids is connected with water-soluble polymers.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, non-naturally encoded amino acids comprises carbonyl, aminooxy, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl.In certain embodiments, on the position of the group that residue 1-5, the 82-90,117-134 and the 169-176 that are selected from by SEQ ID NO:3 (hGH) form, replace non-naturally encoded amino acids.

The present invention also provides medical composition, its comprise pharmaceutically acceptable supporting agent and comprise SEQ ID NO:1,2,3 or any other GH peptide sequence shown in the hGH polypeptide of sequence, wherein at least a amino acid is replaced by non-naturally encoded amino acids.In certain embodiments, non-naturally encoded amino acids comprises sugar moieties.In certain embodiments, water-soluble polymers is connected in polypeptide via sugar moieties.In certain embodiments, connexon, polymkeric substance or bioactive molecules are connected in the hGH polypeptide via sugar moieties.

The present invention also provides the hGH that comprises water-soluble polymers polypeptide, and described water-soluble polymers is connected in single amino acid on the described hGH polypeptide via covalent linkage.In certain embodiments, water-soluble polymers comprises poly-(ethylene glycol) part.In certain embodiments, be covalently attached to the non-naturally encoded amino acids of amino acid in polypeptide, existing of water-soluble polymers.In certain embodiments, non-naturally encoded amino acids 35,92,143 or 145 is substituted in the position.

The invention provides the hGH polypeptide that comprises at least a connexon, polymkeric substance or bioactive molecules, the functional group that wherein said connexon, polymkeric substance or bioactive molecules are incorporated the non-naturally encoded amino acids in the polypeptide into via rrna is connected in polypeptide.In certain embodiments, polypeptide is through single Pegylation.The present invention also provides the hGH polypeptide that comprises the connexon, polymkeric substance or the bioactive molecules that are connected in one or more non-naturally encoded amino acids, and wherein said non-naturally encoded amino acids is to incorporate in the polypeptide via rrna in the preliminary election site.

Definition

Should be appreciated that and the invention is not restricted to described ad hoc approach, experimental program, clone herein, construct body and reagent and therefore can be changed.Should be appreciated that also method used herein is the purpose that only is used to describe specific embodiment, and and be not intended to limit category of the present invention, category of the present invention will only be limited by the claims of enclosing.

Unless context is clearly specified in addition, otherwise as in this paper and the claims of enclosing used singulative " " and " as described in " comprise quoting of plural form.Therefore, for example,, and comprise known its equivalent of those skilled in the art or the like for being cited as of " hGH " for one or more described proteic quoting.

Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art and usually understand identical implication.Though in practice of the present invention or test, can use any method, device and the material similar or equivalent, describe preferred method, device and material now with person described herein.

All open cases mentioned herein and patent all are to be incorporated herein by reference to be used for describing and disclosing purpose, for example construct body and method can be used in combination with invention described herein described in the application case.Only provide open case discussed in this article in the disclosure of the present patent application before the date.There is not content will be interpreted as admitting that the inventor can because of previous invention or any other be former thereby disclose described disclosure in advance herein.

Term " through purifying substantially " mean the hGH polypeptide can be substantially or be not contained in substantially found in the naturally occurring environment of albumen (be blastema, or under the situation of the hGH polypeptide that reorganization produces, be host cell) follow albumen or component interactional usually with it.The hGH polypeptide that can not contain cell material substantially comprise have be less than about 30%, be less than about 25%, be less than about 20%, be less than about 15%, be less than about 10%, be less than about 5%, be less than about 4%, be less than about 3%, be less than about 2% or be less than the proteic protein formulation of contaminative of about 1% (with dry weight basis).When producing hGH polypeptide or its varient by host cell reorganization, albumen can dry cell weight about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% or about 1% or still less exist.When producing hGH polypeptide or its varient by host cell reorganization, about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or the about 1mg/L that albumen can dry cell weight or still less be present in the substratum.Therefore, " through purifying substantially " the hGH polypeptide that is produced by method of the present invention can have at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, purity level at least about 70%, especially at least about 75%, 80%, 85% purity level and more particularly at least about 90% purity level, purity level at least about 95%, purity level at least about 99% or higher, it is by analyzing such as SDS/PAGE, RP-HPLC, the proper method of SEC and capillary electrophoresis is measured.

" recombinant host cell " or " host cell " means the cell that comprises exogenous polynucleotide, and no matter use which kind of method to insert, for example directly absorb to produce recombinant host cell, known other method in transduction, f pairing or the affiliated field.Exogenous polynucleotide can remain the nonconformity carrier of plasmid for example or can be integrated in the host genome.

As used herein, term " medium " (medium or media) comprises any substratum, solution, solid, semisolid or the rigid carrier that can support or contain any host cell, and described host cell comprises bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, Chinese hamster ovary celI or intestinal bacteria and entocyte.Therefore, the medium of the host cell of wherein having grown can be contained in described term, and for example the hGH polypeptide has been secreted into medium wherein, is included in the medium of propagation before or after the step.Damping fluid or the reagent that contains the host cell lysate also can be contained in described term, such as produce in cell at the hGH polypeptide and host cell under dissolving or breaking with the situation that discharges the hGH polypeptide.

Be defined in to go back about albumen folding more employed " reductive agent " herein and keep sulfydryl ortho states under and redox molecule is interior or any compound or the material of intermolecular disulfide bond.Appropriate reductant includes, but is not limited to dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, halfcystine, cysteamine (2-aminoothyl mercaptan) and reduced glutathione.Those of ordinary skill in the field are easy to understand multiple reductive agent and are applicable in the method and composition of the present invention.

Being defined as about albumen folding more employed " oxygenant " herein can be from removing any compound or the material of electronics through the compound of oxidation.Suitable oxygenant comprises (but being not limited to) oxidized glutathione, Gelucystine, cystamine, oxidation dithiothreitol (DTT), oxidation erythritol and oxygen.Those of ordinary skill in the field are easy to understand multiple oxygenant and are applicable in the method for the present invention.

" denaturing agent " used herein is defined as any compound or the material that will cause that the reversible protein folding is opened.The intensity of denaturing agent will be determined by the characteristic and the concentration of specific denaturing agent.Suitable denaturing agent can be the combination of chaotropic agent, sanitising agent, organic solvent, water-miscible solvent, phosphatide or two or more described medicament.Suitable chaotropic agent includes, but is not limited to urea, guanidine and Sodium Thiocyanate 99.The sanitising agent that is suitable for can include, but is not limited to: strong sanitising agent, such as sodium lauryl sulphate or Soxylat A 25-7 (for example Tween or Triton sanitising agent), Sarkosyl; Moderate nonionic sanitising agent (for example digitonin); Moderate cationic sanitising agent, such as N → 2,3-(two oily alkene oxygen bases)-propyl group-N, N, N-trimethyl ammonium; The ionic sanitising agent of moderate (such as cholate or sodium deoxycholate); Or the zwitter-ion sanitising agent, include, but is not limited to sulfonic group trimethyl-glycine (Zwittergent), 3-(3-chloro-acid amide base propyl group) dimethylammonio-1-propane vitriol (CHAPS) and 3-(3-chloro-acid amide base propyl group) dimethylammonio-2-hydroxyl-1-propane sulfonate (CHAPSO).Can use such as acetonitrile, low carbon number alkanol (C especially₂-C₄Alkanol is such as ethanol or Virahol) or low carbon number alkane glycol (C especially₂-C₄The alkane glycol is such as ethylene glycol) organic water-miscible solvent as denaturing agent.Be applicable to that the phosphatide among the present invention can be the phosphatide of natural generation, such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositols; Or synthetic phospholipid derivative or varient, such as two caproyl phosphatidylcholines or two oenanthyl phosphatidylcholines.

" folding again " used herein describes any process, reaction or the method relevant with disulfide linkage, and its polypeptide that will contain disulfide linkage is converted into primary or suitably folding conformation from improper folding or not folding state.

" altogether folding " used herein especially means folding process again, reaction or the method for use at least two peptide species, and described polypeptide is interact with each other and cause folding or improper folding polypeptide is converted into primary suitably folding polypeptide.

As used herein, " tethelin " or " GH " should comprise those have the bioactive polypeptide of at least a human growth hormone and albumen with and the GH analogue, GH is with the merit iso series, the GH simulant, the GH fragment, hybridization GH albumen, fusion rotein oligomer and polymer, homologue, glycosylation pattern varient and mutain, and no matter whether biological activity identical, and further no matter it synthesizes or which kind of method is manufacture method be, no matter include, but is not limited to reorganization (is to produce from cDNA, genomic dna, synthetic DNA still is the nucleic acid of other form), synthetic, change and grow gene and gene activation method.

The hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance contained in term " hGH polypeptide ".For example, exemplary replacement is included in the Methionin replacement of the 41st of primary hGH or the 176th phenylalanine replacement.In some cases, be positioned at the 41st if replace, replacement can be Isoleucine or arginine residues so, if perhaps the position is 176, replacement can be tyrosine residues so.Position F10 can replace through for example A, H or I.Position M14 can replace through for example W, Q or G.Other exemplary replacement comprises any replacement or its combination, includes, but is not limited to:

R167N、D171S、E174S、F176Y、I179T；

R167E、D171S、E174S、F176Y；

F10A、M14W、H18D、H21N；

F10A、M14W、H18D、H21N、R167N、D171S、E174S、F176Y、I179T；

F10A、M14W、H18D、H21N、R167N、D171A、E174S、F176Y、I179T；

F10H、M14G、H18N、H21N；

F10A, M14W, H18D, H21N, R167N, D171A, T175T, I179T; Or

F10I、M14Q、H18E、R167N、D171S、I179T。For example, referring to United States Patent (USP) the 6th, 143, No. 523, it is incorporated herein by reference.

Be described in the exemplary replacement in the multiple amino acids position among the hGH of natural generation, comprise increasing the agonist activity, increasing protease resistant, polypeptide is transformed into the replacement of antagonist etc., and contained by term " hGH polypeptide ".

For example, agonist hGH sequence comprises the hGH sequence that comprises the natural generation of modifying H18D, H21N, R167N, D171S, E174S, I179T.For example referring to United States Patent (USP) the 5th, 849, No. 535, it is incorporated herein by reference.Extra agonist hGH sequence comprises:

H18D、Q22A、F25A、D26A、Q29A、E65A、K168A、E174S；

H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174S; Or

H18D、Q22A、F25A、D26A、Q29A、E65A、K168A、E174A。For example, referring to United States Patent (USP) the 6th, 022, No. 711, it is incorporated herein by reference.The hGH polypeptide that comprises replacement at H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A strengthens at site I place the avidity to the hGH acceptor.For example referring to United States Patent (USP) the 5th, 854, No. 026, it is incorporated herein by reference.HGH sequence with protease resistant of increase includes, but is not limited to comprise the hGH polypeptide of one or more aminoacid replacement in the C-D ring.In certain embodiments, replacement includes, but is not limited to R134D, T135P, K140A and any combination thereof.For example, referring to people such as Alam, (1998) J.Biotechnol.65:183-190.

For example, human growth hormone's antagonist comprises that those have the antagonist that replaces and further comprise following replacement sometimes at G120 (for example G120R, G120K, G120W, G120Y, G120F or G120E): H18A, Q22A, F25A, D26A, Q29A, E65A, K168A, E174A.For example referring to United States Patent (USP) the 6th, 004, No. 931, it is incorporated herein by reference.In certain embodiments, the hGH antagonist comprises at least one replacement making GH serve as among the regional 106-108 of antagonist or the 127-129.For example referring to United States Patent (USP) the 6th, 608, No. 183, it is incorporated herein by reference.In certain embodiments, the hGH antagonist comprises non-naturally encoded amino acids, and described amino acid is connected in existing water-soluble polymers in II land, the site of hGH molecule.In certain embodiments, the hGH polypeptide further comprises following replacement: H18D, H21N, R167N, K168A, D171S, K172R, E174S, I179T, has replacement at G120 simultaneously.(for example referring to United States Patent (USP) the 5th, 849, No. 535).

About GH aminoacid sequence and the GH aminoacid sequence of ripe natural generation and the mutant of natural generation of the natural generation of complete total length, please respectively referring to herein SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.In certain embodiments, hGH polypeptide of the present invention is consistent with any other sequence of SEQ ID NO:1 or SEQ ID NO:2 or SEQ IDNO:3 or growth hormone polypeptides substantially.Discerned the mutant of the natural generation of multiple hGH.These mutant comprise hGH-V (Seeberg, DNA 1:239 (1982); United States Patent (USP) the 4th, 446,235,4,670,393 and 4,665, No. 180, it is incorporated herein by reference) and contain 20-kDa hGH (people such as Kostyo, the Biochem.Biophys.Acta 925:314 (1987) of the residue 32-46 disappearance of hGH (SEQ ID NO:3); Lewis, people such as U., J.Biol.Chem., 253:2679-2687 (1978)).Placental growth hormone is described in Igout, people such as A., Nucleic AcidsRes.17 (10): in 3998 (1989).In addition, reported by transcribing numerous hGH varients that back, translation back, secretion property, metabolism processed-type and other physiological process produce, comprise proteolysis type cracked varient or 2 chain varients (Baumann, G., Endocrine Reviews 12:424 (1991)).Be described in Lewis via the direct-connected hGH dimer of Cys-Cys disulfide linkage, people such as U.J., J.Biol.Chem.252:3697-3702 (1977); Brostedt, P. and Roos, P. is among the Prep.Biochem.19:217-229 (1989).The nucleic acid molecule of coding hGH mutant and mutant hGH polypeptide is well-known, and includes, but is not limited to those in United States Patent (USP) the 5th, 534,617,5,580,723,5,688,666,5,750,373,5,834,250,5,834,598,5,849,535,5,854,026,5,962,411,5,955,346,6,013,478,6,022,711,6,136,563,6,143,523,6,428,954,6,451,561,6, the nucleic acid molecule that discloses in 780, No. 613 and the U.S. Patent Application Publication case 2003/0153003, described document is incorporated herein by reference.

The commercial formulation of hGH is to sell with following title: Humatrope^TM(EliLilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono).

Term " hGH polypeptide " also comprises the steric isomer of hGH of prodrug, polymorph, hydrate, solvate, bioactive fragment, biological activity varient and the natural generation of salt that it is pharmaceutically acceptable and prodrug and described salt, and the agonist of the hGH of natural generation, simulant and antagonist varient and polypeptide syzygy thereof.The syzygy that comprises additional amino acid at N-terminal, C-terminal or two ends is contained by term " hGH polypeptide ".For example, exemplary syzygy include, but is not limited to methionyl tethelin (wherein methionine(Met) is connected in the N-terminal by the hGH of recombinant expressed generation), be used for the purifying purpose syzygy (include, but is not limited to and poly Histidine or affinity epitope merge), with the syzygy of serum albumin binding peptide and with the syzygy of serum protein (such as serum albumin).

The various announcement by polymkeric substance joint or glycosylation with reference to case modified polypeptide.Term " hGH polypeptide " comprises the polypeptide that joins to such as the polymkeric substance of PEG, and can comprise additionally deriving of one or more halfcystines, Methionin or other residue.In addition, the hGH polypeptide can comprise connexon or polymkeric substance, wherein the amino acid that connexon or polymkeric substance engaged can be according to alpha-non-natural amino acid of the present invention, under perhaps can using in the field known technology join natural amino acids coding to, such as with Methionin or halfcystine coupling.

The polymkeric substance of having reported the hGH polypeptide engages.For example referring to United States Patent (USP) the 5th, 849,535,6,136,563 and 6,608, No. 183, it is incorporated herein by reference.United States Patent (USP) the 4th, 904, No. 584 announcements exhaust the polypeptide of Pegylation Methionin, and wherein at least one lysine residue lacks or is replaced by any other amino-acid residue.WO 99/67291 discloses the method that is used for adaptor protein and PEG, wherein lack on the albumen at least one amino-acid residue and be enough to realize with condition that albumen engages under with the PEG contactin.WO 99/03887 discloses the Pegylation varient of the polypeptide that belongs to the tethelin Superfamily, and wherein cysteine residues is replaced by the non-essential amino acid residue that is positioned at the polypeptide designated area.WO 00/26354 discloses the method that produces the glycosylated polypeptides varient, and described polypeptide variants is compared the supersensitivity that causes with reduction with the parent polypeptide that comprises at least one extra glycosylation site.

Term " hGH polypeptide " also comprises the polypeptide of the glycosylation form that is connected with N or is connected with O.The varient that contains single Nucleotide variation also is regarded as the biological activity varient of hGH polypeptide.In addition, also comprise splicing variants.Term " hGH polypeptide " also comprises hGH polypeptide heterodimer, homodimer, heteropolymer or homopolymer, or other bioactive molecule of any other polypeptide, albumen, carbohydrate, polymkeric substance, small molecules, ligand or any kind (its connected by chemical mode or be expressed as fusion rotein) and contain and for example keep bioactive specificity disappearance or other modified polypeptides analogue.

Unless otherwise indicated (that is, and when claim relatively be based on SEQ ID NO:1,3 or during other hGH sequence), otherwise all be based on position among the SEQ ID NO:2 for all references of amino acid position among the hGH as herein described.Be understood by those skilled in the art that, corresponding to SEQ ID NO:1,2,3 or any other GH sequence in the amino acid position of position can be easy to identification in what its hGH molecule in office (such as hGH syzygy, varient, fragment etc.).For example, such as the sequence alignment program of BLAST can be used for comparing with recognition protein in corresponding to SEQ ID NO:1,2,3 or other GH sequence in the specific position of position.Herein about SEQ ID NO:1,2,3 or the described aminoacid replacement of other GH sequence, disappearance or interpolation be also intended to mean replacement, disappearance or interpolation in the correspondence position of known hGH syzygy, varient, fragment etc. in described herein or affiliated field, and it is clearly contained for the present invention.

The hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance contained in term " hGH polypeptide ".HGH polypeptide of the present invention can comprise the modification of one or more natural amino acids and modify together with one or more alpha-non-natural amino acids.Be described in the locational exemplary replacement of multiple amino acids in the hGH polypeptide of natural generation, it includes, but is not limited to regulate one or more the replacement in the biological activity of hGH polypeptide, such as, but not limited to increasing the agonist activity, increase the polypeptide solvability, change polypeptide into antagonist etc., and it is contained by term " hGH polypeptide ".

Human GH antagonist include, but is not limited to 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 have replace or the 1st (being N-terminal) have add or its any combination (SEQ ID NO:2, or SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) antagonist.In certain embodiments, the hGH antagonist comprises at least one replacement that makes GH serve as antagonist in lower area: 1-5 (N-terminal), 6-33 (A spiral), the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-terminal).In other embodiments, the exemplary site of incorporating non-naturally encoded amino acids into comprises the amino terminal region of spiral A and the residue in the part spiral C.In another embodiment, replace G120 with non-naturally encoded amino acids (such as to azido--L-phenylalanine or O-propargyl-L-tyrosine).In other embodiments, with above-mentioned replacement with to make that the hGH polypeptide becomes the extra replacement of hGH antagonist combined.For example, replace non-naturally encoded amino acids one of in position that this paper discerned and introduce at G120 and to replace (for example G120R, G120K, G120W, G120Y, G120F or G120E) simultaneously.In certain embodiments, the hGH antagonist comprise with the receptor binding domain that is present in the hGH molecule in the non-naturally encoded amino acids that is connected of water-soluble polymers.

In certain embodiments, the hGH polypeptide further comprises the bioactive interpolation of adjusting hGH polypeptide, replacement or disappearance.For example, described interpolation, replacement or disappearance can be regulated avidity, adjusting (including, but is not limited to increases or reduce) receptor dimerization effect to the hGH polypeptide receptor, stablize receptor dimer, are regulated circulating half-life, the adjustment of treatment agent transformation period, regulate polypeptide stability, adjusting dosage, adjustment release or biological utilisation, help purifying or improvement or change specific dosing way.Similarly, the hGH polypeptide can comprise protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequence based on avidity (including, but is not limited to FLAG, poly-His, GST etc.) or the molecule (including, but is not limited to vitamin H) through connecting that the improvement polypeptide detects (including, but is not limited to GFP), purifying or further feature.

Heterodimer, homodimer, heteropolymer or the homopolymer that is connected in (including, but is not limited to directly connect via the non-naturally encoded amino acids side chain) identical or different non-naturally encoded amino acids side chain, is connected in natural amino acids coding side chain or connects indirectly via connexon also contained in term " hGH polypeptide ".Exemplary connexon includes, but is not limited to the water-soluble polymers such as poly-(ethylene glycol) or many dextran or polypeptide.

" non-naturally encoded amino acids " means not is the amino acid of 20 kinds of common amino acids wherein a kind of or dried meat Methionin or seleno-cysteine.Other term that can use with the free burial ground for the destitute with term " non-naturally encoded amino acids " is " alpha-non-natural amino acid ", " non-natural generation amino acid " and various band hyphen thereof and not with the variant of hyphen.Term " non-naturally encoded amino acids " also includes, but is not limited to following amino acid: it is to produce by modifying (for example posttranslational modification) natural amino acids coding (including, but is not limited to 20 kinds of common amino acids or dried meat Methionin and seleno-cysteine), but himself and can't help to translate in the natural polypeptide chain of incorporating in the growth of complex body.Described non-natural produces amino acid whose example and includes, but is not limited to N-acetyl glucosamine base-L-Serine, N-acetyl glucosamine base-L-Threonine and O-phosphate tyrosine.

" N-terminal is modified base " means and can be connected in the aminoterminal any molecule of polypeptide.Similarly, " C-terminal is modified base " means any molecule that can be connected in the polypeptide C-terminal.End modified base includes, but is not limited to various water-soluble polymerss, peptide or albumen (such as serum albumin) or other increases the part of the serum half-life of peptide.

Term " functional group ", " active part ", " activation base ", " leaving group ", " reaction site ", " chemical reaction base " and " chemical reactivity part " is in affiliated field and be definable molecular moiety or the unit that is used to mean uniqueness herein.Described term has a synonym in chemical field, and is used for indication in this article and carries out some function or active and to the reactive molecular moiety of other molecule tool.

Term " bonding " or " connexon " are used to mean the group that forms usually as the result of chemical reaction or key in this article and are generally covalent bonding.To the bonding of hydrolysis-stable mean be bonded in the water stable substantially and in prolonging period (perhaps or even endless) (do not including, but is not limited under physiological condition) reaction under the applicable pH value with water.To bonding hydrolytically unstable or degradable mean be bonded in the water or the aqueous solution (for example comprising blood) in be degradable.Unsettled or the degradable bonding of enzymolysis is meaned bonding can be by one or more enzyme liberating.Such as in the affiliated field understanding, PEG and related polymer can comprise degradable bonding in the connexon group between one or more in polymer backbone or among the terminal functional group of polymer backbone and polymer molecule.For example, by with the formed ester bond of pure radical reaction on PEG carboxylic acid or activated PEG carboxylic acid and the biologically active agent under physiological condition usually hydrolysis with release medicine.The bonding of other hydrolyzable degraded includes, but is not limited to: the carbonic ether bonding; By amine and aldehyde reaction and the imine bound that produces; By the pure phosphoric acid ester bonding that forms with the phosphate groups reaction; Hydrazone bonding for the reaction product of hydrazides and aldehyde; Be the acetal bonding of aldehyde with the reaction product of alcohol; Be the former ester linkage of formate with the reaction product of alcohol; By the peptide linkage that includes, but is not limited to form at carboxyl such as the amine groups of the polymer ends of PEG and peptide; With the oligonucleotide bonding that forms by the 5 ' hydroxyl that includes, but is not limited at the phosphoramidite group of polymer ends and oligonucleotide.

Term " bioactive molecules ", " biologically-active moiety " or " biologically active agent " mean any physics that can influence biological organism or any material of biochemical characteristic when using in this article, described organism includes, but is not limited to virus, bacterium, fungi, plant, animal and human's class.Specific, bioactive molecules includes, but is not limited to be intended to be used in the mankind or other animal diagnosis, healing, alleviation, treatment or preventing disease or be used to strengthen the health of the mankind or animal or any material of mental status in addition as used herein.The example of bioactive molecules includes, but is not limited to peptide, albumen, enzyme, small-molecule drug, dyestuff, lipid, nucleosides, oligonucleotide, cell, virus, liposome, particulate and micelle.Be applicable to that biologically active agent classification of the present invention includes, but is not limited to microbiotic, mycocide, antiseptic-germicide, antiphlogistic, antineoplastic agent, cardiovascalar agent, antianxiety agent, hormone, somatomedin, steroid medicament or the like.

" double functional copolymer " mean comprise two can with other parts (including, but is not limited to amino acid side group) specific reaction to form the covalently or non-covalently polymkeric substance that separates the functional group of bonding.Have one and can can be used for forming the joiner that comprises first biologically active components, difunctionality connexon and second biologically active components with the difunctionality connexon of the group of radical reaction on second biological components with the functional group of radical reaction on the particular organisms active ingredient and another.The many programs and the connexon molecule that are used for all cpds is connected in peptide are known.For example referring to No. the 188th, 256, European patent application, United States Patent (USP) the 4th, 671,958,4,659,839,4,414,148,4,699,784,4,680,338,4,569,789 and 4,589, No. 071, it is incorporated herein by reference." polyfunctional poly compound " mean comprise two or more can with other parts (including, but is not limited to amino acid side group) specific reaction to form the covalently or non-covalently polymkeric substance that separates the functional group of bonding.

When substituting group was illustrated by its conventional chemical formula of writing from left to right, it was contained comparably and writes the chemically consistent substituting group that structure obtains, for example structure-CH by turning left from the right side₂O-is equivalent to structure-OCH₂-.

Term " substituting group " includes, but is not limited to " non-interfering substituting group "." non-interfering substituting group " generates the substituting group of stable compound for those.Suitable non-interfering substituting group or group include, but is not limited to halogen, C₁-C₁₀Alkyl, C₂-C₁₀Thiazolinyl, C₂-C₁₀Alkynyl, C₁-C₁₀Alkoxyl group, C₁-C₁₂Aralkyl, C₁-C₁₂Alkaryl, C₃-C₁₂Cycloalkyl, C₃-C₁₂Cycloalkenyl group, phenyl, be substituted phenyl, tolyl, xylyl, xenyl, C₂-C₁₂Alkoxyalkyl, C₂-C₁₂Alkoxy aryl, C₇-C₁₂Aryloxy alkyl, C₇-C₁₂Oxygen aryl, C₁-C₆Alkyl sulphinyl, C₁-C₁₀Alkyl sulphonyl ,-(CH₂)_m-O-(C₁-C₁₀Alkyl) (wherein m is 1 to 8), aryl, be substituted aryl, be substituted alkoxyl group, fluoroalkyl, heterocyclic radical, be substituted heterocyclic radical, 4-nitro alkyl ,-NO₂,-CN ,-NRC (O)-(C₁-C₁₀Alkyl) ,-C (O)-(C₁-C₁₀Alkyl), C₂-C₁₀Alkyl alkylthio base ,-C (O) O-(C₁-C₁₀Alkyl) ,-OH ,-SO₂,=S ,-COOH ,-NR₂, carbonyl ,-C (O)-(C₁-C₁₀Alkyl)-CF₃,-C (O)-CF₃,-C (O) NR₂,-(C₁-C₁₀Aryl)-S-(C₆-C₁₀Aryl) ,-C (O)-(C₁-C₁₀Aryl) ,-(CH₂)_m-O-((CH₂)_m-O-(C₁-C₁₀Alkyl) (wherein each m is 1 to 8) ,-C (O) NR₂,-C (S) NR₂,-SO₂NR₂,-NRC (O) NR₂,-NRC (S) NR₂, its salt or the like.Each R used herein is H, alkyl or is substituted alkyl, aryl or is substituted aryl, aralkyl or alkaryl.

Term " halogen " comprises fluorine, chlorine, iodine and bromine.

Unless otherwise indicated, otherwise term " alkyl " self or mean straight or branched or cyclic hydrocarbon group or its combination during as another substituent part, it can be saturated fully, single or how unsaturated, and can comprise that having the appointment carbon atom number (is C₁-C₁₀Mean 1 to 10 carbon) divalence and multivalence group.The example of saturated hydrocarbyl includes, but is not limited to such as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl; The group of the homologue of n-pentyl, n-hexyl, n-heptyl, n-octyl and isomer or the like.Unsaturated alkyl is for having one or more pairs key or triple-linked alkyl.The example of unsaturated alkyl includes, but is not limited to vinyl, 2-propenyl, butenyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), ethynyl, 1-and 3-proyl, 3-butynyl and Geng Gao carbon number homologue and isomer.Unless otherwise, otherwise term " alkyl " also means and comprises those alkyl derivatives of specific definition hereinafter, such as " assorted alkyl ".The alkyl that only is limited to alkyl is called as " homotype alkyl ".

Term " alkylidene group " self or mean during as another substituent part be derived from alkane divalent group (such as but not limited to structure-CH₂CH₂-and-CH₂CH₂CH₂CH₂-), and comprise that further those are described to the group of " assorted alkylidene group " hereinafter.Alkyl (or alkylidene group) has 1 to 24 carbon atom usually, wherein those have 10 or still less the group of carbon atom be preferred in the present invention." low carbon number alkyl " or " low carbon number alkylidene group " is than short-chain alkyl or alkylidene group, and it has 8 or carbon atom still less usually.

Term " alkoxyl group ", " alkylamino " and " alkylthio " (or thioalkoxy group) use with its conventional sense, and mean those and be connected to alkyl on the molecule remainder via Sauerstoffatom, amino or sulphur atom.

Unless otherwise indicated, otherwise term " assorted alkyl " self or mean stable straight or branched or cyclic hydrocarbon group or its combination during with another term combination, it is made up of the heteroatoms that the carbon atom that specifies number and at least one are selected from the group that is made up of O, N, Si and S, and wherein nitrogen and sulphur atom can according to circumstances can be according to circumstances through quaternized through oxidation and nitrogen heteroatom.Heteroatoms O, N, S and Si can place any interior location of assorted alkyl or place alkyl to be connected in the position of molecule remainder.Example includes, but is not limited to-CH₂-CH₂-O-CH₃,-CH₂-CH₂-NH-CH₃,-CH₂-CH₂-N (CH₃)-CH₃,-CH₂-S-CH₂-CH₃,-CH₂-CH₂-S (O)-CH₃,-CH₂-CH₂-S (O)₂-CH₃,-CH=CH-O-CH₃,-Si (CH₃)₃,-CH₂-CH=N-OCH₃With-CH=CH-N (CH₃)-CH₃Two heteroatomss can be successive at the most, for example-and CH₂-NH-OCH₃With-CH₂-O-Si (CH₃)₃Similarly, term " assorted alkylidene group " self or mean the divalent group that is derived from assorted alkyl during as another substituent part (such as but not limited to-CH₂-CH₂-S-CH₂-CH₂-and-CH₂-S-CH₂-CH₂-NH-CH₂-).For assorted alkylidene group, identical or different heteroatoms also can occupy one or two chain end (including, but is not limited to alkylene oxide group, alkylene dioxo base, alkylidene amino, alkylidene group diamino, aminooxy alkylidene group or the like).In addition, for alkylidene group was connected base with assorted alkylidene group for, the direction that the formula of connection base is write did not show the orientation that connects base.For example, formula-C (O)₂R '-representative-C (O)₂R '-and-R ' C (O)₂-.

Unless otherwise indicated, otherwise term " cycloalkyl " and " Heterocyclylalkyl " self or represent the ring-type variant of " alkyl " and " alkyl of mixing " during with the combination of other term respectively.Therefore, cycloalkyl or Heterocyclylalkyl comprise that saturated and unsaturated ring key closes.In addition, for Heterocyclylalkyl, heteroatoms can occupy the position that heterocycle is connected in the molecule remainder.The example of cycloalkyl includes, but is not limited to cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl or the like.The example of Heterocyclylalkyl includes, but is not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl or the like.In addition, dicyclo and tricyclic structure contained in described term.Similarly, term " heterocycle alkylidene group " self or mean divalent group during as another substituent part, and term " ring alkylidene group " self or mean divalent group during as another substituent part derived from cycloalkyl derived from Heterocyclylalkyl.

As used herein, term " water-soluble polymers " means any polymkeric substance that dissolves in the water-based solvent.The bonding of water-soluble polymers and hGH polypeptide can cause changing, the serum half-life that includes, but is not limited to increase or regulates or with respect to the immunogenicity of the therapeutical agent transformation period that increases without modified forms or regulate, adjusting, the physics association feature of adjusting (such as assembling and polymer formation), the receptors bind of change and the receptor dimerization or the poly effect of change.Water-soluble polymers may have or may not have himself biological activity.Suitable polymers includes, but is not limited to polyoxyethylene glycol, polyoxyethylene glycol propionic aldehyde, its single C₁-C₁₀Alkoxyl group or aryloxy derivative (are described in United States Patent (USP) the 5th, 252, in No. 714, it is incorporated herein by reference), mono methoxy-polyoxyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, the divinyl ether MALEIC ANHYDRIDE, N-(2-hydroxypropyl)-Methacrylamide, dextran, glucan derivative (comprising T 500), polypropylene glycol, polyoxytrimethylene/ethylene oxide copolymer, polyoxy ethylization polyvalent alcohol, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, Mierocrystalline cellulose and derivatived cellulose (including, but is not limited to methylcellulose gum and carboxymethyl cellulose), starch and starch derivative, polypeptide, polyalkylene glycol and derivative thereof, the multipolymer of polyalkylene glycol and derivative thereof, polyvinyl ethyl ether and alpha-beta-poly-[(2-hydroxyethyl)-DL-l-asparagine or the like or its mixture.The example of described water-soluble polymers includes, but is not limited to polyoxyethylene glycol and serum albumin.

As used herein, term " polyalkylene glycol " or " poly-(aklylene glycol) " mean polyoxyethylene glycol (poly-(ethylene glycol)), polypropylene glycol, polytetramethylene glycol and derivative thereof.Straight chain and branched chain polymer and the molecular-weight average between 0.1kDa and 100kDa contained in term " polyalkylene glycol ".For example, other exemplary embodiments is recited in the commercial supplier catalogue, such as Shearwater Corporation catalogue " Polyethylene Glycol and Derivatives forBiomedical Applications " (2001).

Unless otherwise indicated, otherwise term " aryl " means polyunsaturated aromatic hydrocarbon substituting group, and it can be monocycle or is fused to together or covalently bound many rings (being preferably 1 to 3 ring).Term " heteroaryl " means and contains 1 to 4 heteroatomic aryl that is selected from N, O and S (or ring), and wherein nitrogen and sulphur atom be according to circumstances through oxidation, and nitrogen-atoms is according to circumstances through quaternized.Heteroaryl can be connected in the molecule remainder via heteroatoms.The limiting examples of aryl and heteroaryl comprises phenyl, the 1-naphthyl, the 2-naphthyl, the 4-xenyl, the 1-pyrryl, the 2-pyrryl, the 3-pyrryl, the 3-pyrazolyl, the 2-imidazolyl, the 4-imidazolyl, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridyl, the 3-pyridyl, the 4-pyridyl, the 2-pyrimidyl, the 4-pyrimidyl, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl-, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.The substituting group that is used for above-mentioned each aryl and heteroaryl ring system is to be selected from the group that is made up of the substituting group accepted hereinafter described.

For for simplicity, term " aryl " is when comprising aryl and heteroaryl ring as defined above when being used in combination with other term (including, but is not limited to aryloxy, fragrant sulphur oxygen base, arylalkyl).Therefore, term " arylalkyl " mean comprise those wherein aryl be connected in the group (including, but is not limited to phenmethyl, styroyl, pyridylmethyl or the like) of alkyl, it comprise those wherein carbon atom (including, but is not limited to methylene radical) by for example Sauerstoffatom metathetical alkyl (including, but is not limited to phenoxymethyl, 2-pyridyloxy methyl, 3-(1-naphthyloxy) propyl group or the like).

Each above-mentioned term (including, but is not limited to " alkyl ", " assorted alkyl ", " aryl " and " heteroaryl ") all means and comprises the appointment group that is substituted and is unsubstituted form.Hereinafter be provided for the exemplary substituting group of every type of group.

The substituting group that is used for alkyl and assorted alkyl (comprising that those are commonly called alkylidene group, thiazolinyl, assorted alkylidene group, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) can be and is selected from but is not limited to one or more of multiple group of following group :-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R  ,-OC (O) R ' ,-C (O) R ' ,-CO₂R ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R  ,-NR " C (O)₂R ' ,-NR-C (NR ' R " R )=NR " " ,-NR-C (NR ' R ")=NR  ,-S (O) R ' ,-S (O)₂R ' ,-S (O)₂NR ' R " ,-NRSO₂R ' ,-CN and-NO₂, number is 0 in the scope of (2m '+1), and wherein m ' is the overall number of carbon atom in the described group.R ', R ", R  and R " " the assorted alkyl that means hydrogen independently of one another, is substituted or is unsubstituted, the aryl that is substituted or is unsubstituted (include, but is not limited to replace aryl), alkyl, alkoxyl group or the thioalkoxy group or the arylalkyl that are substituted or are unsubstituted through 1-3 halogen.When compound of the present invention comprised an above R group, for example, each R group all was independent selection, when having R ', R ", R  and R " " when one of group is above, each R ', R ", R  and R " " the group situation is the same.As R ' and R " when being connected in identical nitrogen-atoms, it can make up to form 5,6 or 7 yuan of rings with nitrogen-atoms.For example ,-and NR ' R " mean and include, but is not limited to 1-pyrrolidyl and 4-morpholinyl.According to substituent above the discussion, be understood by those skilled in the art that term " alkyl " means and comprise following group: it comprises the carbon atom that is attached to the group outside the dehydrogenation base, (includes, but is not limited to-CF such as haloalkyl₃With-CH₂CF₃) and acyl group (include, but is not limited to-C (O) CH₃,-C (O) CF₃,-C (O) CH₂OCH₃Or the like).

Be similar to for the described substituting group of alkyl, being used for the substituting group of aryl and heteroaryl can be different and be to be selected from but to be not limited to: halogen ,-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-SiR ' R " R  ,-OC (O) R ' ,-C (O) R ' ,-CO₂R ' ,-CONR ' R " ,-OC (O) NR ' R " ,-NR " C (O) R ' ,-NR '-C (O) NR " R  ,-NR " C (O)₂R ' ,-NR-C (NR ' R " R )=NR " " ,-NR-C (NR ' R ")=NR  ,-S (O) R ' ,-S (O)₂R ' ,-S (O)₂NR ' R " ,-NRSO₂R ' ,-CN and-NO₂,-R ' ,-N₃,-CH (Ph)₂, fluorine (C₁-C₄) alkoxyl group and fluorine (C₁-C₄) alkyl, number is 0 on the aromatic ring system in the scope of open valent overall number, and wherein R ', R ", R  and R " " be independently be selected from halogen, alkyl, mix alkyl, aryl and heteroaryl.When compound of the present invention comprised an above R group, for example, each R group all was independent selection, when having R ', R ", R  and R " " when one of group is above, each R ', R ", R  and R " " the group situation is identical.

As used herein, term " serum half-life of adjusting " means modified bioactive molecules with respect to positivity or the negativity change of its non-modified forms on circulating half-life.Serum half-life is to measure by each time point blood-sample withdrawal after the bioactive molecules that comes into operation and the concentration of measuring molecule described in each sample.The dependency of serum-concentration and time allows to calculate serum half-life.The serum half-life that increases has the serum half-life at least about 2 times ideally, but littler increase may be suitable for, and for example wherein it is facilitated gratifying dosage regimen or avoids toxic effect.In certain embodiments, described increase at least about 3 times, at least about 5 times or at least about 10 times.

Term " the therapeutical agent transformation period of adjusting " means the modified bioactive molecules of treatment significant quantity with respect to positivity or the negativity change of its non-modified forms on the transformation period as used herein.The therapeutical agent transformation period is to measure by the pharmacokinetics and/or the pharmacodynamic profiles of each point in time measurement molecule after dispensing.The therapeutical agent transformation period that increases facilitates specific useful dosage regimen, specific useful total dose ideally or avoids improper effect.In certain embodiments, the therapeutical agent transformation period of increase is combining or another parameter of the effect of non-decorating molecule or mechanism increases or reduction and causing by the modified molecule of the effectiveness, increase or the reduction that increase and its target.

Term " through separating " is when being used for nucleic acid or albumen, and its expression nucleic acid does not contain other cellular component relevant with its native state substantially with albumen.It can be homogeneous state.Can be drying or partial desiccation state or be the solution form through separate substance, it includes, but is not limited to the aqueous solution.Purity and homogeneity typically use such as the technique of analytical chemistry of polyacrylamide gel electrophoresis or high performance liquid chromatography and measure.For the albumen of existing essential substance in the preparation purified substantially.Specific, be to separate through isolated genes from surrounding gene both sides and the coding proteic open reading frame except that genes involved.Term " purified " expression nucleic acid or albumen reach a band substantially in running gel.Specifically, it means nucleic acid or albumen and has at least 85% purity, at least 90% purity, at least 95% purity, at least 99% purity or high purity more.

Term " nucleic acid " means deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Unless specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in described term, described analogue have the binding characteristic that is similar to reference nucleic acid and carry out metabolism in the mode of the Nucleotide that is similar to natural generation.Unless other specific limited, otherwise described term also means oligonucleotide analogs, it comprises PNA (peptide nucleic acid(PNA)), used DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters or the like) in antisense technology.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and clear and definite specified sequence.Specific, can realize that through mixing sequence that base and/or Hypoxanthine deoxyriboside residue replace degenerate codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing one of them or selected more than one (or all) codon the 3rd; People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); People such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).

Term " polypeptide ", " peptide " and " albumen " exchange in this article and use to mean the polymkeric substance of amino-acid residue.That is, be equally applicable to describe peptide and describe albumen at the description of polypeptide, and vice versa.Described term is applicable to natural generation aminoacid polymers and one of them or the aminoacid polymers that above amino-acid residue is a non-naturally encoded amino acids.As used herein, the amino acid chain of any length contained in described term, and it comprises full-length proteins (being antigen), and wherein amino-acid residue connects via the covalency peptide bond.

Term " amino acid " means natural generation and non-natural produces amino acid, and brings into play the amino acid analogue and the amino acid analog agent of function to be similar to the amino acid whose mode of natural generation.Natural amino acids coding is 20 kinds of common amino acids (L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) and dried meat Methionin and seleno-cysteine.Amino acid analogue means the compound with Essential Chemistry structure identical with natural generation amino acid, and promptly α carbon combines with hydrogen, carboxyl, amino and R group, such as homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.Described analogue has modified R group (such as nor-leucine) or modified peptide backbone, but keeps the Essential Chemistry structure identical with natural generation amino acid.

Can amino acid whose common known trigram symbol or quote amino acid with the one-letter symbol of recommending by IUPAC-IUB commission on Biochemical nomenclature.Common acceptable single-letter code that equally can Nucleotide is quoted Nucleotide.

" through the conservative varient of modifying " is applicable to amino acid and nucleotide sequence.For specific nucleic acid sequence, " through the conservative varient of modifying " means following nucleic acid: aminoacid sequence or its amplifying nucleic acid that its coding is consistent or consistent basically do not become consistent basically sequence with amino acid sequence encode.Because the degeneracy of genetic code, so any given albumen of the nucleic acid encoding of many function unanimities.For example, codon GCA, GCC, GCG and GCU coded amino acid L-Ala all.Therefore, in each position with codon explanation L-Ala, codon all can not change encoded polypeptide through changing into described any corresponding codon.Described nucleic acid variant is " a reticent variant ", and it is a kind of conservative variant of modifying of warp of kind.Each nucleotide sequence of coded polypeptide is also described the reticent variant of each possible nucleic acid herein.Those skilled in the art will realize that each codon in the nucleic acid is (except that AUG, it is unique password that is generally used for methionine(Met), and except that TGG, it is unique password that is generally used for tryptophane) all can modified molecule with the systematic function unanimity.Therefore, the reticent variant of each of nucleic acid encoding implies in each described sequence.

About aminoacid sequence, those skilled in the art will realize that indivedual replacements, disappearance or interpolation to nucleic acid, peptide, polypeptide or protein sequence (its change, add or lack single amino acid in the coded sequence or the amino acid of little per-cent) be " through the conservative varient of modifying ", wherein said change cause amino acid through chemically similarly amino acid replace.It is well-known in affiliated field that functionally similar amino acid whose conservative replacement table is provided.The conservative varient of modifying of described warp is outside homologue and allelic scope between polymorphic varient of the present invention, kind but it is not foreclosed.

Below 8 groups contain separately another amino acid be the conservative amino acid that replaces:

1) L-Ala (A), glycine (G);

2) aspartic acid (D), L-glutamic acid (E);

3) l-asparagine (N), glutamine (Q);

4) arginine (R), Methionin (K);

5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V);

6) phenylalanine (F), tyrosine (Y), tryptophane (W);

7) Serine (S), Threonine (Y); With

8) halfcystine (C), methionine(Met) (M)

(for example referring to Creighton, Proteins:Structures and Molecular Properties (W H Freeman ﹠amp; Co.; The 2nd edition (in December, 1993)).

Term in the context of two or more nucleic acid or peptide sequence " unanimity " or " consistence " per-cent mean two or more sequence or subsequence identical.When using one of following sequence comparison algorithm or on comparison window or measured designated area, relatively reaching when comparing maximum correspondence by manual comparison and range estimation, have identical amino-acid residue or the per-cent of Nucleotide (60% consistence of promptly on the designated area, having an appointment as infructescence, be according to circumstances about 65%, about 70%, about 75%, about 80%, about 85%, about 90% or about 95% consistence), sequence is " substantially consistent " so.This definition also means the complementation of cycle tests.On at least about the zone of 50 amino acid or length of nucleotides or on the zone of 75-100 amino acid or length of nucleotides, can have consistence, perhaps when not specifying, in whole sequence or polynucleotide or polypeptide, can there be consistence.

For sequence comparatively speaking, a common sequence is served as reference sequences, and cycle tests compares with it.When using sequence comparison algorithm, with cycle tests and reference sequences input computer, then specify the subsequence coordinate if desired, and specified sequence algorithm routine parameter.The default program parameter can be used, perhaps alternate parameter can be specified.Sequence comparison algorithm calculates the sequence identity per-cent of cycle tests with respect to reference sequences based on program parameter then.

" comparison window " comprises quoting sections with one of close position number of being selected from the group that is made up of following number as used herein: 20 to 600, be generally about 50 to about 200, be more typically about 100 to about 150, wherein can after sequence and the best comparison of the reference sequences of the close position with similar number it be compared.The comparison method that is used for the sequence of comparison is well-known in affiliated field.Can include, but is not limited to local homology's algorithm with Smith and Waterman (1970) Adv.Appl.Math.2:482c, homology alignment algorithm with Needleman and Wunsch (1970) J.Mol.Biol.48:443, research with the similar degree method of Pearson and Lipman (1988) Proc.Nat ' I.Acad.Sci.USA 85:2444, (WisconsinGenetics Software Package is implemented in computerize with described algorithm, Genetics Computer Group, 575 Science Dr., Madison, GAP among the WI, BESTFIT, FASTA and TFASTA) or be used for the best comparison of the sequence of comparison with manual comparison and range estimation (for example referring to people such as Ausubel, Current Protocols in Molecular Biology (1995 supplementary issue)).

An example that is applicable to the algorithm of measuring sequence identity per-cent and sequence similar degree is BLAST and BLAST2.0 algorithm, it is described in people such as Altschul respectively, (1977) people such as Nuc.Acids Res.25:3389-3402 and Altschul are among (1990) J.Mol.Biol.215:403-410.Being used to carry out the software that BLAST analyzes can obtain by NCBI (National Center for Biotechnology Information) publicly.BLAST algorithm parameter W, T and X determine the sensitivity and the speed of comparison.BLASTN program (being used for nucleotide sequence) is used following default parameters: word length (W) is 11, and expected value (E) is 10, M=5, and N=-4 also compares two chains.For aminoacid sequence, the BLASTP program is used following default parameters: word length be 3 and expected value (E) be 10, score matrix (referring to Henikoff and Henikoff (1989) Proc.Natl with BLOSUM62, Acad.Sci.USA89:10915), comparison value (B) is 50, expected value (E) is 10, M=5, two chains of N=-4 and comparison.When carrying out the BLAST algorithm, close " low complex degree " screening procedure usually.

The BLAST algorithm is also carried out the statistical study (for example referring to Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of similar degree between two sequences.A kind of similar degree method of masurement that the BLAST algorithm is provided is minimum summation probability (P (N)), and it provides the indication to the probability that will take place accidentally between two Nucleotide or the aminoacid sequence to mate.For example, if in the comparison of test nucleic acid and reference nucleic acid minimum summation probability less than about 0.2, be more preferably less than about 0.01 and most preferably less than about 0.001, think that so nucleic acid and reference sequences are similar.

Phrase " selectivity (or specificity) hybridizes to " means when having specific nucleotide sequence in compounding mixture, and molecule only combines, forms duplex or hybridization (including, but is not limited to total cell or library DNA or RNA) with described sequence under rigorous hybridization conditions.

Phrase " rigorous hybridization conditions " means known low ionic strength and pyritous condition in affiliated field.Usually, under rigorous condition, probe will hybridize on its target subsequences in the compounding mixture (including, but is not limited to total cell or library DNA or RNA) of nucleic acid, but not hybridize on other sequence in the compounding mixture.Rigorous condition be sequence dependent and in varying environment with different.Long sequence specific hybrid under comparatively high temps.Detailed guidance for nucleic acid hybridization is found in Tijssen, Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes is in " Overview of principles ofhybridization and thestrategy of nucleic acid assays " (1993).Rigorous condition is selected as being lower than the heat fusion joint (T of distinguished sequence under regulation ionic strength pH usually_m) about 5-10 ℃.T_mFor under equilibrium state 50% with target complementary probe hybridization during to target sequence residing temperature (under specify ion intensity, pH and nucleic acid concentration) (because of the excessive existence of target sequence, so at T_m50% probe is occupied under equilibrium state down).Rigorous condition can be following condition: wherein be lower than about 1.0M Na ion concentration pH7.0 to 8.3 time salt concn, be generally about 0.01 to 1.0M Na ion concentration (or other salt), and temperature is at least about 30 ℃ for short probe (including, but is not limited to 10 to 50 Nucleotide), and is at least about 60 ℃ for long probe (including, but is not limited to greater than 50 Nucleotide).Rigorous condition also can realize such as the destabilizing agent of methane amide by adding.For selectivity or specific hybrid, positive signal can be the background hybridization of twice at least, is 10 times of background hybridizations according to circumstances.Exemplary rigorous hybridization conditions can be as follows: 50% methane amide, and 5 * SSC and 1%SDS cultivate down at 42 ℃; Or 5 * SSC, 1%SDS cultivates down at 65 ℃, washs in 0.2 * SSC and washs in 0.1%SDS under 65 ℃.Described washing can be carried out 5,15,30,60,120 minutes or the longer time.

As used herein, term " eukaryote " means and belongs to kind of an organism that is generation eucaryon field, such as animal (including, but is not limited to Mammals, insect, Reptilia, birds etc.), ciliate, plant (including, but is not limited to monocotyledons, dicotyledons, algae etc.), fungi, yeast, flagellate, microsporidium, protobiont etc.

As used herein, term " non-eukaryote " means non-eucaryon organism.For example, non-eucaryon organism can belong to eubacterium and (include but not limited to intestinal bacteria, thermophilic bacterium (Thermus thermophilus), bacstearothermophilus (Bacillus stearothermophilus), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putidas (Pseudomonas putida) etc.) planting is the generation field, or archeobacteria (includes but not limited to Methanococcus jannaschii (Methanococcus jannaschii), thermophilic methagen (Methanobacterium thermoautotrophicum), halophilic bacterium (Halobacterium) (having a liking for richly endowed bacterium of salt (Haloferax volcanii) and halophilic bacterium kind NRC-1) such as Wo Shi, have a liking for ultrahigh-temperature sulfate radical reduction archeobacteria (Archaeoglobus fulgidus), fierce fireball bacterium (Pyrococcus furiosus), Huo Shi fireball bacterium (Pyrococcushorikoshii), hyperthermophilic archaeon strains (Aeuropyrum pemix) etc.) planting is the generation field.

Term used herein " person under inspection " means the animal into treatment, observation or experimental subjects, is preferably Mammals, most preferably is the mankind.

Term used herein " significant quantity " means the amount of (modified) non-natural amino acid polypeptides that is come into operation, and it alleviates in the symptom of the disease for the treatment of, illness or symptom one or more to a certain extent.The composition that contains (modified) as herein described non-natural amino acid polypeptides that can come into operation is treated to be used for prevention type, enhancement type and/or therapeutic type.

Term " enhancing " means to be renderd a service or is increasing on the time length or the prolongation effect of wanting.Therefore, about strengthening the effect of therapeutical agent, term " enhancing " means increases or prolongs the ability of other therapeutical agent to the effect of system on effectiveness or time length." enhancing significant quantity " used herein means the amount that is enough to strengthen the effect of another therapeutical agent in want system.When using in the patient, the amount that can be effective to this purposes will depend on the seriousness of disease, symptom or illness and the course of disease, previous therapy, patient health situation and to the reaction of medicine and treatment doctor's judgement.

Term used herein " modified " means and have posttranslational modification on polypeptide.It is modified according to circumstances that form " (modified) " term means the polypeptide of being discussed, and promptly the polypeptide of being discussed can be modified or not modified.

Term " through posttranslational modification " and " modified " mean after natural or alpha-non-natural amino acid have been incorporated in the polypeptide chain described amino acid whose any modification.Only with example it is described, described term is contained the interior modification of common translation body, the interior modification of translation back body and is translated the external modification in back.

In the prevention type was used, the composition that will contain (modified) non-natural amino acid polypeptides came into operation to easy trouble specified disease, symptom or illness or the patient of the risk of suffering from specified disease, symptom or illness is arranged.Described amount is defined as " prevention significant quantity ".In this purposes, accurately amount also depends on patient health situation, weight or the like.The those skilled in the art fully understands with normal experiment (for example dosage constantly promotes clinical trial) and determines described prevention significant quantity.

Term " through protection " means and have " protecting group " or the part that stops the chemical reactivity functional group to react under special reaction condition.Protecting group will look protect the type of chemical reaction base and change.For example, if the chemical reaction base is amine or hydrazides, protecting group can be selected from tert-butoxycarbonyl (t-Boc) and 9-fluorenyl methoxy carbonyl (Fmoc) so.If the chemical reaction base is a mercaptan, protecting group can be adjacent pyridyl disulfide so.If the chemical reaction base is such as the carboxylic acid of butyric acid or propionic acid or hydroxyl, protecting group can be phenmethyl or such as the alkyl of methyl, ethyl or the tertiary butyl so.Known other protecting group also can be used for method and composition as herein described in the affiliated field.

Only with example it is described, END CAPPED GROUP/protecting group can be selected from:

Other protecting group is described in Greene and Wuts, Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, its mode of quoting in full is incorporated herein.

In therapeutic type is used, to be enough to cure or to the amount of the symptom of small part retardance disease, symptom or illness, the composition that will contain (modified) non-natural amino acid polypeptides comes into operation to the patient who suffers from described disease, illness or symptom.Described amount is defined as " treatment significant quantity ", and will depend on the seriousness of disease, symptom or illness and the course of disease, previous therapy, patient health situation and to the reaction of medicine and treatment doctor's judgement.The those skilled in the art fully understands with normal experiment (for example dosage constantly promotes clinical trial) and determines described treatment significant quantity.

Term " treatment " is in order to mean the treatment of prevention type and/or therapeutic type.

Unless otherwise, otherwise use the ordinary method that is appreciated by one of skill in the art that, such as mass spectroscopy, NMR, HPLC, albumen chemistry, biological chemistry, recombinant DNA technology and pharmacology.

Description of drawings

Fig. 1 shows the proteic universal architecture of four-helix bundle.

Fig. 2 shows the universal architecture of four-helix bundle albumen tethelin (GH).

Fig. 3 shows the universal architecture of four-helix bundle albumen erythropoietin (EPO).

Fig. 4 shows the universal architecture of four-helix bundle albumen interferon (IFN α-2).

Fig. 5 shows the universal architecture of four-helix bundle protein granules population of cells stimulating factor (G-CSF).

Fig. 6 shows Ku Masi indigo plant (Coomassie blue) dyeing SDS-PAGE, and its proof comprises the expression of non-naturally encoded amino acids to the hGH of acetyl phenyl alanine on following each position: Y35, F92, Y111, G131, R134, K140, Y143 or K145.

Fig. 7 picture A and B show the hGH (picture B) that comprises non-naturally encoded amino acids and wild-type hGH (picture A) biological activity to the IM9 cell.

Fig. 8 shows the blue dyeing of Ku Masi SDS-PAGE, and its proof comprises by PEG (5,20 and 30kDa) with the non-naturally encoded amino acids covalent bonding and through the generation of the hGH of the non-naturally encoded amino acids of Pegylation.

Fig. 9 shows the biological activity of the hGH of the various Pegylation forms that comprise non-naturally encoded amino acids to the IM9 cell.

Figure 10 picture A describes to have the primary structure (from people such as Becker, the figure of Biotechnol Appl Biochem. (1988) 10 (4): 326-337 reorganization) of specifying the trypsinase cracking site and replacing the hGH of F92pAF with the alpha-non-natural amino acid of arrow explanation.Figure 10 picture B shows the overlapping trypsinase collection of illustrative plates of following material: by non-naturally encoded amino acid whose hGH polypeptide produces through comprising of Pegylation peptide (through the A of mark), by comprising peptide (through the B of mark) that non-naturally encoded amino acid whose hGH polypeptide produces and by the peptide (through the C of mark) of WHO rhGH generation.Figure 10 picture C is the enlarged view of thepeak value 9 of picture B.

Figure 11 picture A and picture B show that the blue dyeing of the Ku Masi SDS-PAGE of purified PEG-hGH polypeptide analyzes.

Figure 12 shows the biological activity of hGH dimer molecule to the IM9 cell.

Figure 13 picture A shows that measurement has the IM-9 calibrating data of the hGH antagonist of G120R replacement to the phosphorylation of pSTAT5.Figure 13 picture B displaying is measured in same position (G120) and is incorporated the IM-9 calibrating data of the hGH polypeptide of alpha-non-natural amino acid to the phosphorylation of pSTAT5 into.

Figure 14 shows that the dimer that shows the hGH antagonist shown in Figure 13 picture B also lacks biological activity in the IM-9 calibrating.

Figure 15 show through the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation with without the rat of the hGH polypeptide of Pegylation in the comparison diagram of serum half-life.

Figure 16 shows the comparison diagram of serum half-life in the rat of the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation.

Figure 17 shows the comparison diagram of serum half-life in the rat of the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation.Rat is administered once with 2.1mg/kg.

Figure 18 picture A is illustrated in the effect that after the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation (position 35,92) rat body weight is increased of throwing with single dose.Figure 18 picture B be illustrated in throw with single dose after the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation (position 35,92) to the effect of circulating plasma IGF-1 content.Figure 18 picture C is illustrated in the effect that after the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation (position 92,134,145,131,143) rat body weight is increased of throwing with single dose.Figure 18 screen D be illustrated in throw with single dose after the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation (position 92,134,145,131,143) to the effect of circulating plasma IGF-1 content.Figure 18 picture E shows the comparison diagram of serum half-life in the rat of the hGH polypeptide that comprises non-naturally encoded amino acids of Pegylation (position 92,134,145,131,143).

Embodiment

I. introduce

The invention provides the hGH molecule that comprises at least one alpha-non-natural amino acid.In certain embodiments of the present invention, the hGH polypeptide with at least a alpha-non-natural amino acid comprises at least one posttranslational modification.In one embodiment, described at least one posttranslational modification comprises uses the known chemical process that is applicable to specific reactive group of those of ordinary skill in the field, the molecule that will comprise second reactive group is connected in the alpha-non-natural amino acid that at least one comprises first reactive group, and the described molecule that comprises second reactive group includes, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, cytotoxic compound, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, inhibition Yeast Nucleic Acid, biomaterial, nano particle, spin labeling, fluorophore, the containing metal part, radioactive segment, novel functional group, with the covalently or non-covalently interactional group of other molecule, the light enclosure portion, but the part of photoisomerization, vitamin H, biotin derivative, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, prolong side chain, the sugar that is connected with carbon, redox active agent, amino thioic acid sulfoacid, the toxicity part, isotope-labeled part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable label, above-mentioned or any other of small molecules wanted any combination of compound or material.For example, first reactive group be alkynyl part (include, but is not limited to alpha-non-natural amino acid to alkynes propoxy-phenylalanine in, wherein propargyl is also sometimes referred to as acetylene moiety), and second reactive group is the nitrine part, and use [3+2] cycloaddition chemical process.In another embodiment, first reactive group is nitrine part (includes, but is not limited to alpha-non-natural amino acid to azido--L-phenylalanine in), and second reactive group is the alkynyl part.In some embodiment of modified hGH polypeptide of the present invention, use comprises at least one alpha-non-natural amino acid (including, but is not limited to contain ketone group functional group's alpha-non-natural amino acid) of at least one posttranslational modification, and wherein said at least one posttranslational modification comprises sugar moieties.In certain embodiments, in eukaryotic cell or non-eukaryotic cell, carry out posttranslational modification in the body.

In certain embodiments, albumen comprises at least one by the posttranslational modification of carrying out in a kind of host cell body, and wherein said posttranslational modification be can't help another host cell type usually and carried out.In certain embodiments, albumen comprises at least one by the posttranslational modification of carrying out in the eukaryotic cell body, and wherein said posttranslational modification be can't help non-eukaryotic cell usually and carried out.The example of posttranslational modification includes, but is not limited to acetylize, acidylate, lipid-modified, palmitoylation, palmitinic acid addition, phosphorylation, the glycolipid bonding is modified or the like.In one embodiment, posttranslational modification comprises and via GlcNAc-l-asparagine key oligosaccharides is connected in l-asparagine (including, but is not limited to wherein, oligosaccharides comprises (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc or the like).In another embodiment, posttranslational modification comprises via GalNAc-Serine, GalNAc-Threonine, GlcNAc-Serine or GlcNAc-Threonine bonding oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc etc.) is connected in Serine or Threonine.In certain embodiments, albumen of the present invention or polypeptide can comprise that secretion or positioning sequence, epitope label, FLAG label, poly are histidine-tagged, GST syzygy or the like.

Associated protein or polypeptide can contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten or more alpha-non-natural amino acids.Alpha-non-natural amino acid can be identical or different, and for example 1,2,3,4,5,6,7,8,9,10 in albumen or more different loci can comprise 1,2,3,4,5,6,7,8,9,10 or how different alpha-non-natural amino acids.In certain embodiments, at least a (but being less than all) in the specific amino acids that exists in the protein variant of natural generation replaces through alpha-non-natural amino acid.

The invention provides based on GH supergene family member, specificly be the method and composition of hGH, it comprises at least a non-naturally encoded amino acids.To can allow to use the joint chemistry among at least a non-naturally encoded amino acids introducing GH supergene family member, it comprises and (including, but is not limited to) one or more non-naturally encoded amino acids specificity chemical reactions, simultaneously not with the 20 seed amino acids reaction of common generation.In certain embodiments, the GH supergene family member who comprises non-naturally encoded amino acids is connected in water-soluble polymers via the side chain of non-naturally encoded amino acids, such as polyoxyethylene glycol (PEG).The invention provides the high efficiency method that is used for PEG derivatives selectively modified protein, it comprises incorporates non-genomic amino acids coding selectivity in the albumen of replying the selection codon into, and modify described amino acid with the PEG derivative of suitable reactivity subsequently, described amino acid includes, but is not limited to those amino acid that contains in 20 kinds of natural amino acid of incorporating into undiscovered functional group or substituting group (including, but is not limited to ketone, nitrine or acetylene moiety).After incorporating into, amino acid side chain then can those of ordinary skill in the field are known to be applicable to that the particular functional base or the substituent chemical process that exist in the natural amino acids coding are modified by utilizing.Multiple known chemical process is applicable to that the present invention is to incorporate water-soluble polymers in the albumen into.Described method includes, but is not limited to respectively the Huisgen[3+2 with (including, but is not limited to) acetylene or azido derivant] and cycloaddition reaction (for example referring to Padwa, A.Comprehensive Organic Synthesis, the 4th volume, (1991) Trost, B.M. compiles, Pergamon, Oxford, 1069-1109 page or leaf; And Huisgen, R.1.3-Dipolar Cvcloaddition Chemistry. (1984) Padwa, A. compiles, Wiley, New York, 1-176 page or leaf).

Because Huisgen[3+2] the cycloaddition method comprises cycloaddition reaction rather than nucleophilic substitution reaction, so selective modification albumen that can be high.Can under room temperature, react under the aqueous conditions by Cu (I) salt of catalytic amount being added in the reaction mixture with the fabulous locational choice (1,4＞1,5).For example referring to people such as Tornoe, (2002)Org.Chem.67:3057-3064; With people such as Rostovtsev, (2002)Angew.Chem.Int.Ed.41:2596-2599; And WO03/101972.Can almost comprise any molecule via the molecule that [3+2] cycloaddition reaction adds on the albumen of the present invention with suitable functional group or substituting group (including, but is not limited to azido-or acetylene-derivative).These molecules can add on the alpha-non-natural amino acid (including, but is not limited to alkynes propoxy-phenylalanine) with acetylene group respectively or have on the alpha-non-natural amino acid (including, but is not limited to the triazobenzene L-Ala) of azido-.

By Huisgen[3+2] five-ring that produces of cycloaddition is not reversible usually in the reductibility environment and is stable to hydrolysis in over a long time in aqueous environment.Therefore, the physics of multiple material and chemical feature can be modified with active PEG derivative of the present invention under required aqueous conditions.The more important thing is because nitrine and acetylene moiety for be each other specific (and for example not with the amino acid of 20 kinds of genes encodings in any reaction), so can be in one or more specificity sites with high selective modification albumen.

The present invention also provides the water-soluble of PEG derivative and stability to hydrolysis derivative and the relevant hydrophilic polymer with one or more acetylene or nitrine part.The PEG polymer derivant that contains acetylene moiety is a high selectivity for introducing for the nitrine part coupling of replying in the albumen of selecting codon with selectivity.Similarly, the PEG polymer derivant that contains nitrine part is a high selectivity for introducing for the acetylene moiety coupling of replying in the albumen of selecting codon with selectivity.

More particularly, the nitrine part is including but not limited to the derivative of alkyl azide, aromatic yl azide and these trinitride.The derivative of alkyl and aromatic yl azide can comprise other substituting group, as long as the acetylene specific reaction is kept.Acetylene moiety comprises alkyl and aryl ethane and derivative separately.The derivative of alkyl and aryl ethane can comprise other substituting group, as long as the nitrine specific reaction is kept.

The invention provides and have multiple functional group, the material of substituting group or part and the joiner of other material, described other material includes, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, the derivative of polyoxyethylene glycol, the photocrosslinking agent, cytotoxic compound, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, inhibition Yeast Nucleic Acid, biomaterial, nano particle, spin labeling, fluorophore, the containing metal part, radioactive segment, novel functional group, with the covalently or non-covalently interactional group of other molecule, the light enclosure portion, but the part of photoisomerization, vitamin H, biotin derivative, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, prolong side chain, the sugar that is connected with carbon, redox active agent, amino thioic acid sulfoacid, the toxicity part, isotope-labeled part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable label, any combination or any other of small molecules or above-mentioned substance are wanted compound or material.The present invention also comprises the joiner of material with nitrine or acetylene moiety and the PEG polymer derivant with corresponding acetylene or nitrine part.For example, the PEG polymkeric substance that contains nitrine part can contain coupling on the position of the non-genomic amino acids coding with acetylene functional group with bioactive molecules in albumen.PEG and bioactive molecules coupling via bonding include, but is not limited to Huisgen[3+2] the cycloaddition product.

Confirmed that in affiliated field PEG can be used for the surface of modified biological material (for example referring to United States Patent (USP) the 6th, 610, No. 281 and Mehvar, R., J.Pharmaceut.Sci., 3 (1): 125-136 (2000), it is incorporated herein by reference).The present invention also comprises biomaterial, its comprise surface with one or more reactive nitrine or acetylene site and via Huisgen[3+2] cycloaddition bonding and surperficial coupling of the present invention contain one or more in nitrine or the acetylene polymer.Biomaterial and other material also can be via the bondings except that nitrine or acetylene bonding and through nitrine or the coupling of acetylene activatory polymer derivant, such as via the bonding that comprises carboxylic acid, amine, alcohol or thiol moiety, to stay nitrine or the acetylene moiety that can be used for afterreaction.

The present invention includes the synthetic method that contains nitrine and acetylene polymer of the present invention.Under the situation that contains nitrine PEG derivative, nitrine directly bond to the carbon atom of polymkeric substance.Perhaps, be connected in conventional activated polymkeric substance so that resulting polymers has the nitrine part at its end, so can prepare and contain nitrine PEG derivative by the linking agent that will at one end have the nitrine part.Under the situation that contains acetylene PEG derivative, acetylene directly bond to the carbon atom of polymkeric substance.Perhaps, be connected in conventional activated polymkeric substance so that resulting polymers has acetylene moiety at its end, so can prepare and contain acetylene PEG derivative by the linking agent that will at one end have acetylene moiety.

More particularly, under the situation that contains nitrine PEG derivative, the water-soluble polymers experience reaction with at least one activity hydroxy part is to produce the polymkeric substance that is substituted that has reactive higher part (such as methanesulfonate, trifluoro ethyl sulfonic acid root, tosylate or halogen leaving group) on it.The preparation and the purposes that contain the PEG derivative of alkylsulfonyl acid halide, halogen atom and other leaving group are well-known for the those skilled in the art.Gained be substituted polymkeric substance experience subsequently reaction with in polymer ends with the higher part of nitrine part substitution reaction.Perhaps, the water-soluble polymers with at least one active nucleophilicity or electrophilicity part reacts with the linking agent that at one end has nitrine, so that form covalent linkage between PEG polymkeric substance and linking agent, and nitrine partly is positioned at polymer ends.The nucleophilicity and the electrophilicity part that comprise amine, mercaptan, hydrazides, hydrazine, alcohol, carboxylate radical, aldehyde, ketone, thioesters or the like are well-known for the those skilled in the art.

More particularly, under the situation that contains acetylene PEG derivative, the water-soluble polymers experience reaction with at least one activity hydroxy part is to replace halogen or other activated leaving group from the presoma that contains acetylene moiety.Perhaps, the water-soluble polymers with at least one active nucleophilicity or electrophilicity part reacts with the linking agent that at one end has acetylene, so that form covalent linkage between PEG polymkeric substance and linking agent, and acetylene moiety is positioned at polymer ends.The use of the halogen part in the context of the organic synthesis of PEG derivative and preparation and use, activated leaving group, nucleophilicity and electrophilicity part is well-known for the implementer in affiliated field.

The present invention also is provided for selective modification albumen other material is added to the method on the modified albumen, includes, but is not limited to the water-soluble polymers such as PEG that contains nitrine or acetylene moiety and PEG derivative.The PEG derivative that contains nitrine and acetylene can be used for modifying wherein biocompatibility, stability, solvability and the surface of immunogenicity shortage tool importance and the characteristic of molecule, and connection PEG derivative and proteic mode than previously known mode selectivity is higher in the affiliated field are provided simultaneously.

II. tethelin supergene family

Following albumen comprises those albumen by the genes encoding of tethelin (GH) supergene family (Bazan, F., Immunology Today 11:350-354 (1991); Bazan, J.F.Science 257:410-411 (1992); Mott, H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Hile, J.N., SIGNALLING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS (1996)): tethelin, prolactin, galactagogin, erythropoietin (EPO), thrombopoietin (TPO), IL-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), interferon-alpha, interferon-, the ε Interferon, rabbit, IFN-, omega interferon, the τ Interferon, rabbit, granulocyte colony stimulating factor (G-CSF), granular leukocyte macrophage group stimulating factor (GM-CSF), scavenger cell group stimulating factor (M-CSF) and heart nutrient substance-1 (CT-1) (" GH supergene family ").Be expected at and discern the extra member of this gene family via gene clone and order-checking future.The member of GH supergene family has similar secondary and tertiary structure, though it has limited amino acid or consensus dna sequence usually.Total constitutional features allows easily to discern the newcomer of gene family and uses alpha-non-natural amino acid method and composition as herein described similarly.Consider the degree of the structural homology among the GH supergene family member, can use the present invention that non-naturally encoded amino acids is incorporated among any member of GH supergene family.Each member of this protein family comprises four-helix bundle, and it is the universal architecture of that shown in Figure 1.The universal architecture of family member hGH, EPO, IFN α-2 and G-CSF is showed in respectively among Fig. 2,3,4 and 5.

Determined the structure of various kinds of cell hormone by X-ray diffraction and NMR, and it shows the surprising observation to the GH structure, though lack remarkable primary sequence homology, described cytohormone comprises G-CSF (people such as Zink, FEBSLett.314:435 (1992); People such as Zink, Biochemistry 33:8453 (1994); People such as Hill, Proc.Natl.Acad.Sci.USA 90:5167 (1993)), GM-CSF (Diederichs, people such as K., Science 154:1779-1782 (1991); People such as Walter, J.Mol.Biol.224:1075-1085 (1992)), IL-2 (Bazan, J.F.Science 257:410-411 (1992); McKay, D.B.Science 257:412 (1992)), IL-4 (people such as Redfield, Biochemistry 30:11029-11035 (1991); People such as Powers, Science 256:1673-1677 (1992)) and IL-5 (people such as Milburn, Nature 363:172-176 (1993)).Based on modeling and other research, IFN is regarded as member (people such as Lee, the J.Growth hormone Cytokine Res.15:341 (1995) of this family; People such as Murgolo, Proteins 17:62 (1993); People such as Radhakrishnan, Structure 4:1453 (1996); People such as Klaus, J.Mol.Biol.274:661 (1997)).Bring out research based on modeling and sudden change, EPO is regarded as member (people such as Boissel, the J.Biol.Chem.268:15983-15993 (1993) of this family; People such as Wen, J.Biol.Chem.269:22839-22846 (1994)).Think that now all above-mentioned cytohormones and somatomedin form a very big gene family.

Except that total similar secondary and tertiary structure, the member of this family has following characteristic: it must close cell surface receptor with signal path in the activating cells by oligomerization.Some GH family members that include, but is not limited to GH and EPO are in conjunction with the acceptor of single type and make it form homodimer.Other family member who includes, but is not limited to IL-2, IL-4 and IL-6 is in conjunction with the acceptor of more than one types and make acceptor form heterodimer or high-order aggregate (people such as Davis, (1993), Science 260:1805-1808 more; People such as Paonessa, (1995), EMBO is J.14:1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995)).Sudden change is brought out research and is showed, as GH, described other cytohormone and somatomedin contain a plurality of receptor binding sites (being generally two) also successively in conjunction with its homoreceptor (Mot and Campbell, Current Opinion in Structural Biology 5:114-121 (1995); People such as Matthews, (1996) Proc.Natl.Acad.Set USA 93:9471-9476).As GH, the principal recipient binding site that is used for described other family member mainly is present in four α spirals and A-B ring.The specific amino acid that participates in receptors bind in the helical bundle is different in the family member.Great majority and the interactional cell surface receptor of GH supergene family member be structurally associated and form second largest multigene family.For example referring to United States Patent (USP) the 6th, 608, No. 183, it is incorporated herein by reference.

Usually tend to not be involved in the receptors bind for the ring that engages the α spiral from the common conclusions that each member's of GH supergene family mutation research obtains.Specific, be nonessential as if at the short-and-medium B-C ring of great majority (if not all) family member to receptors bind.For this reason, the B-C ring can replace through non-naturally encoded amino acids as herein described in GH supergene family member.A-B ring, C-D ring (with the interferoid/IL-10 member's of GH supergene family D-E ring) also can produce aminoacid replacement through non-natural.Contiguous spiral A and also tend to not be involved in the receptors bind away from the amino acid of final spiral, and also can be and be used to introduce non-natural and produce amino acid whose site.In certain embodiments, any position of non-naturally encoded amino acids in ring structure is substituted, and includes, but is not limited to preceding 1,2,3,4,5,6,7 or the amino acids place more of A-B, B-C, C-D or D-E ring.In certain embodiments, one or more non-naturally encoded amino acids are at back 1,2,3,4,5,6,7 or more be substituted in the amino acids of A-B, B-C, C-D or D-E ring.

Some the GH family member who includes, but is not limited to EPO, IL-2, IL-3, IL-4, IL-6, G-CSF, GM-CSF, TPO, IL-10, IL-12p35, IL-13, IL-15 and interferon-is contained the sugar that is connected and/or is connected with O with N.Glycosylation site in the albumen almost is present in the ring zone exclusively and is not present in the α helical bundle.Because the ring zone is not involved in the receptors bind and because it is the site that is used for covalently bound glycosyl usually,, it is used for non-natural is produced the suitable site that aminoacid replacement is introduced albumen so can be.The amino acid that comprises the glycosylation site that is connected with O with N in the albumen can be and is used for the site that non-natural produces aminoacid replacement, exposes because these amino acid are the surface.Therefore, the native protein tolerable is connected in proteic huge glycosyl in these site, and glycosylation site tends to away from receptor binding site.

Find the extra member of GH supergene family probably in future.The newcomer that can discern the GH supergene family via the area of computer aided formula secondary and the tertiary structure analysis of institute's predicted protein sequence.The member of GH supergene family has four or five both sexes spirals that engage via non-helical amino acid (ring zone) usually.Albumen can contain the hydrophobicity signal sequence to promote the secretion from cell at its N-terminal.The described GH supergene family member who found afterwards also is included among the present invention.

Therefore, provide for the description of tethelin supergene family being used for illustration purpose, and it only is an example and not as the restriction of the category of method as herein described, composition, strategy and technology.In addition, in the application's case, quoting of GH, IFN, G-CSF and EPO polypeptide is intended to use the example of generic term as any member of GH supergene family.Therefore, should be appreciated that, comprise this paper person of itemizing about hGH, hIFN, hG-CSF or hEPO polypeptide or proteic modification as herein described and chemical any member that can be applied to the GH supergene family comparably.

III. be used for general recombinant nucleic acid method of the present invention

In numerous embodiment of the present invention, recombination method separates with using, clone and the nucleic acid that changes the relevant hGH polypeptide of coding usually.Described embodiment is used for (including, but is not limited to) protein expression or is used to be derived from during the generation of varient, derivative, expression cassette or other sequence of hGH polypeptide.In certain embodiments, the series of operations of code book invention polypeptide is connected in allogeneic promoter.The generation of GH for example is described in United States Patent (USP) the 4th, 601 in the separation of hGH and the host cell, and 980,4,604,359,4,634,677,4,658,021,4,898,830,5,424,199,5,795,745,5,854,026,5,849,535,6,004,931,6,022,711,6,143,523 and 6,608, in No. 183, it is incorporated herein by reference.

Can come composite coding to comprise the nucleotide sequence of the hGH polypeptide of non-naturally encoded amino acids based on the aminoacid sequence of parent polypeptide, it includes, but is not limited to have the aminoacid sequence shown in the SEQ ID NO:2 (hGH), and changes nucleotide sequence then so that realize the introducing (promptly incorporate into or replace) of related amino acid residue or remove (i.e. disappearance or replacement).Can bring out modified nucleotide sequence routinely by rite-directed mutagenesis according to conventional methods.Perhaps, nucleotide sequence can come by the chemosynthesis preparation, include, but is not limited to by use oligonucleotide synthesizer (wherein oligonucleotide is based on the amino acid sequence of polypeptide design of wanting), and preferably select those to produce codon favourable in the host cell of recombinant polypeptide therein.For example, but composite coding want polypeptide part several small oligonucleotides and by PCR, joint or engage chain reaction and assemble.For example referring to people such as Barany, Proc.Natl.Acad.Sci.88:189-193 (1991); United States Patent (USP) the 6th, 521, No. 427, it is incorporated herein by reference.

The present invention utilizes the routine techniques in the genetic recombination field.The base text that discloses useful in the present invention general method comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition .2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); With Current Protocolsin Molecular Biology (people such as Ausubel compiles, 1994).

The general text of describing molecular biotechnology comprises Berger and Kimmel,Guide to Molecular Cloning;Techniques, Methods in Enzymology,The 152nd volume, Academic Press, Inc., San Diego, CA (Berger); People such as Sambrook,Molecular Cloning-A Laboratory Manual (the 2nd edition), the1-3 volume .Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") andCurrent Protocols in Molecular Biology, people such as F.M.Ausubel compile, Current Protocols, a jointventure between Greene Publishing Associates, Inc.and John Wiley ﹠amp; Sons, Lie, (augmenting in 1999) (" Ausubel ").The sudden change of these textual descriptions is brought out, carrier use, promotor and many other relevant propositions, and described proposition is to produce about (including, but is not limited to) to comprise and be used to produce the gene that comprises alpha-non-natural amino acid, quadrature tRNA, quadrature synthetic enzyme and the proteic selection codon of paired thereof.

Use various types of sudden changes to bring out in the present invention, include, but is not limited to produce the tRNA library, produce the synthetic enzyme library, produce in the selection codon insertion associated protein or polypeptide of selecting codon, the alpha-non-natural amino acid of will encoding to be used for various purposes.It includes, but is not limited to fix a point that random point mutation brings out, homologous recombination, DNA resets or other recurrence sudden change is brought out method, the chimeric dna mutation of constructing, use the sudden change that contains the uridylic template to bring out, bring out, modify through thiophosphatephosphorothioate at the sudden change of oligonucleotide and brought out, uses the sudden change of interval duplex DNA to bring out or the like or its any combination.Other suitable method comprises a mispairing reparation, use the sudden change of rectification of defects type host strain to bring out, limit select and restriction purifying, deletion mutantion are brought out, with the synthetic sudden change of carrying out of total gene bring out, bifilar fracture reparation or the like.Including, but is not limited to relate to chimeric sudden change of constructing body brings out also and is included among the present invention.In one embodiment, molecule that can natural generation or through changing or instructing sudden change to bring out through the Given information of the molecule of the natural generation of sudden change, described information comprises (but being not limited to) sequence, sequence comparison, physical features, crystalline structure or the like.

These programs of text of being found and case description herein.Extraneous information is found in the open case of being quoted in the following document and reference: people such as Ling, and Approaches to DNA mutagenesis:an overview,AnalBiochem.254 (2): 157-178 (1997); People such as Dale, Oligonucleotide-directed random mutagenesisusing the phosphorothioate method,Methods Mol.Biol.57:369-374 (1996); Smith, In vitromutagenesis,Ann.Rev.Genet.19:423-462 (1985); Botstein ﹠amp; Shortle, Strategies andapplications of in vitro mutagenesis,Science229:1193-1201 (1985); Carter, Site-directedmutagenesis,Biochem.J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis,Nucleic Acids ﹠amp; Molecular Biology(D.M.J. compiles, SpringerVerlag, Berlin) (1987) for Eckstein, F. and Lilley; Kunkel, Rapid and efficient site-specific mutagenesis withoutphenotypic selection,Proc.Natl.Acad.Sci.USA82:488-492 (1985); People such as Kunkel, Rapid andefficient site-specific mutagenesis without phenotypic selection,Methods in Enzymol.154,367-382 (1987); People such as Bass, Mutant Trp repressors with new DNA-binding specificities,Science242:240-245 (1988);Methods in Enzvmol.100:468-500 (1983);Methods inEnzvmol.154:329-350 (1987); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis usingMl 3-derived vectors:an efficient and general procedure for the production of pointmutations in any DNA fragmentNucleic Acids Res.10:6487-6500 (1982); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors,MethodsIn Enzvmol.100:468-500 (1983); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis:asimple method using two oligonucleotide primers and a single-stranded DNA template,Methods in Enzvmol.154:329-350 (1987); People such as Taylor, The use ofphosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA,Nucl.Acids Res.13:8749-8764 (1985); People such as Taylor, The rapid generation ofoligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA,Nucl.Acids Res.13:8765-8787 (1985); Nakamaye ﹠amp; Eckstein, Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis,Nucl.Acids Res.14:9679-9698 (1986); People such as Sayers, Y-T Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis,Nucl.Acids Res.16:791-802 (1988); People such as Sayers, Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restriction endonucleases in the presenceof ethidium bromide, (1988)Nucl.Acids Res.16:803-814; People such as Kramer, The gapped duplexDNA approach to oligonucleotide-directed mutation construction,Nucl.Acids Res.12:9441-9456 (1984); Kramer ﹠amp; Fritz Oligonucleotide-directed construction of mutations viagapped duplex DNA,Methods in Enzvmol.154:350-367 (1987); People such as Kramer, Improvedenzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations,Nucl.Acids Res.16:7207 (1988); People such as Fritz, Oligonucleotide-directed construction of mutations:a gapped duplex DNA procedure withoutenzymatic reactions in vitro,Nucl.Acids Res.16:6987-6999 (1988); People such as Kramer, PointMismatch Repair,Cell38:879-887 (1984); People such as Carter, Improved oligonucleotidesite-directed mutagenesis using M13 vectors,Nucl.Acids Res.13:4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors,Methods inEnzymol.154:382-403 (1987); Eglitedarzadeh ﹠amp; Henikoff, Use of oligonucleotides togenerate large deletions,Nucl.Acids Res.14:5115 (1986); People such as Wells, Importance ofhydrogen-bond formation in stabilizing the transition state of subtilisin,Phil.Trans.R.Soc.Lond.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning of a gene codingfor the ribonuclease S protein,Science223:1299-1301 (1984); Sakamar and Khorana, Totalsynthesis and expression of a gene for the a-subunit of bovine rod outer segment guaninenucleotide-binding protein (transducin),Nucl.Acids Res.14:6361-6372 (1988); People such as Wells, Cassette mutagenesis:an efficient method for generation of multiple mutations at definedsites,Gene34:315-323 (1985); People such as Grundstrom, Oligonucleotide-directed mutagenesis bymicroscale ' shot-gun ' gene synthesis,Nucl.Acids Res.13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli:amethod for site-specific mutagenesis,Proc.Natl.Acad.Sci.USA, 83:7177-7181 (1986); Arnold, Protein engineering for unusual environments,Current Opinion in Biotechnology4:450-455 (1993); People such as Sieber, Nature Biotechnology, 19:456-460 (2001); W.P.C.Stemmer,Nature370,389-91 (1994); And I.A.Lorimer, I.Pastan,Nucleic Acids Res.23,3067-8 (1995).Extra detailed description for many aforesaid methods is found inMethods in EnzymologyIn the 154th volume, it is also described and is used for the suitable control that the trouble-shooting problem of method is brought out in various sudden changes.

The present invention also relates to be used for via quadrature tRNA/RS to the eukaryotic host cell of incorporating alpha-non-natural amino acid in the body into, non-eukaryotic host cell and organism.With polynucleotide of the present invention or comprise that the body of constructing of polynucleotide of the present invention carries out genetically engineered (including, but is not limited to conversion, transduction or transfection) to host cell, the described body of constructing includes, but is not limited to carrier of the present invention, and it for example can be cloning vector or expression vector.Carrier for example can be plasmid, bacterium, virus, naked polynucleotide or engages the form of polynucleotide.With standard method carrier is introduced in cell and/or the microorganism, described method comprise electroporation (people such as From,Proc.Natl.Acad.Sci.USA82,5824 (1985)), with viral vector infection, with in bead or particulate matrix or have on the surface nucleic acid small-particle high speed ballistic penetration (people such as Klein,Nature327,70-73 (1987)).

Host cell through engineering design can be cultivated in conventional nutritional medium, and described substratum is in due course through regulating to be used for the activity such as screening step, activation promotor or selection transformant.These cells can be cultivated commentaries on classics according to circumstances and grow in the gene organism.Other the suitable reference that is used for (including, but is not limited to) cellular segregation and cultivation (for example being used for separate nucleic acid subsequently) comprises Freshney (1994)Culture of Animal Cells, a Manual of BasicTechnique, the 3rd edition, Wiley-Liss, New York and the reference of wherein quoting; People such as Payne, (1992)Plant Cell and Tissue Culture in Liquid SystemsJohn Wiley ﹠amp; Sons, Inc.New York, NY; Gamborg and Phillips (volume) (1995)Plant Cell.Tissue and Organ CultureFundamental MethodsSpringer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (volume)The Handbook of Microbiological Media(1993) CRC Press, Boca Raton, FL.

Can obtain several target nucleic acid is introduced well-known method in the cell, wherein any method all can be used for the present invention.These methods comprise: with recipient cell and the fusion of bacterium protoplastis, the electroporation that contain DNA, projectile bombardment with virus vector (hereinafter further discussing) injection etc.Bacterial cell can be used for increasing and contains the number that DNA of the present invention constructs the plasmid of body.Make bacterial growth arrive logarithmic phase, and can under the plasmid (for example referring to Sambrook) in the known several different methods separation of bacterial in the field.In addition, can buy many test kits to be used for from the bacteria purification plasmid (for example referring to available from Pharmacia BiotechEasyPrep^TMAnd FlexiPrep^TM: available from the StrataClean of Stratagene^TMWith QIAprep available from Qiagen^TM).Through separate and the plasmid of purifying with after further handle to produce other plasmid, be used for transfectional cell or incorporate related vector into to infect organism.Typical carriers contains transcribes and translation termination, transcribes and translation initiation sequence and be applicable to the promotor of regulation and control specific objective expression of nucleic acids.Carrier comprise according to circumstances contain at least one independently the terminator sequence general expression cassette, allow eukaryote or prokaryotic organism or duplicate the sequence (including, but is not limited to shuttle vectors) of expression cassette among both and be used for prokaryotic organism and the selective marker of eukaryote system.Carrier is applicable at prokaryotic cell prokaryocyte, eukaryotic cell or is preferably and duplicates among both and integrate.Referring to Giliman ﹠amp; Smith,Gene8:81 (1979); People such as Roberts,Nature, 328:731 (1987); Schneider, people such as B.,Protein Expr.Purif.6435:10 (1995); Ausubel, Sambrook, Berger (all as above).For example, provide the bacterium that is applicable to the clone and the catalogue of phage, for example publish by ATCC by ATCCThe ATCC Catalogue of Bacteria andBacteriophage(1992) people's (volume) such as Gherna.Be used to check order, clone and molecular biological others and the potential theoretical extra Basic application of considering also be found in people such as Watson, (1992)Recombinant DNA Second EditionScientific American Books is among the NY.In addition, any substantially nucleic acid (and is almost any nucleic acid through mark, standard is the criteria of right and wrong also) all can any routine or standard from multiple commercial source order, such as MidlandCertified Reagent Company (Midland, TX, can on World Wide Web mcrc.com, obtain), The GreatAmerican Gene Company (Ramona, CA, can on World Wide Web genco.com, obtain), ExpressGen Inc. (Chicago, IL, can on World Wide Web expressgen.com, obtain), (Alameda is CA) with many other sources for Operon Technologies Inc..

Select codon

The genetic code frame of selection codon expansion protein biology combination mechanism of the present invention.For example, select codon to include, but is not limited to three unique base codons, nonsense codon (, including, but is not limited to amber codon (UAG) or opal codon (UGA)), non-natural codon, four or more polybase base codon, rare codon or the like such as terminator codon.Those of ordinary skill in the field be easy to understand can introduce the number range of the selection codon in the gene of wanting very wide, include, but is not limited in the single polynucleotide of coding at least a portion hGH polypeptide for one or more, two or more, more than three, 4,5,6,7,8,9,10 or more.

In one embodiment, described method comprises that the selection codon that uses to terminator codon is incorporating in the eukaryotic cell in the alpha-non-natural amino acid body.For example, produce the O-tRNA of identification terminator codon (including, but is not limited to UAG), and wanted the amino acidylate of O-RS of alpha-non-natural amino acid to have.This O-tRNA also can't help host's aminoacyl-tRNA synthetase of natural generation and discerns.Conventional rite-directed mutagenesis brings out the related locus that is used in the related polypeptide and introduces terminator codon (including, but is not limited to TAG).For example referring to Sayers, people such as J.R., (1988), 5 ', 3 ' Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis.Nucleic Acids Res,791-802.When making up in the nucleic acid body with O-RS, O-tRNA and coding related polypeptide,, incorporate alpha-non-natural amino acid into to obtain containing the polypeptide of alpha-non-natural amino acid at specified location in response to the UAG codon.

Can under the situation that does not significantly upset eukaryotic host cell, carry out incorporating alpha-non-natural amino acid in the body.For example, because depend on competition between O-tRNA (include, but is not limited to amber and suppress sub-tRNA) and eukaryotic releasing factor (including, but is not limited to eRF) (it is attached to terminator codon and peptide of causing in the growth discharges from rrna), so can regulate inhibition efficient by (including, but is not limited to) increase O-tRNA and/or the expression level that suppresses sub-tRNA for the inhibition efficient of UAG codon.

Select codon also to comprise the codon of prolongation, it includes, but is not limited to four or polybase base codon more, such as four, five, six or polybase base codon more.The example of four base codons includes, but is not limited to AGGA, CUAG, UAGA, CCCU or the like.The example of five base codons includes, but is not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC or the like.Feature of the present invention comprises the codon that suppresses to use prolongation based on frameshit.Four or more polybase base codon can (include, but is not limited to) one or more alpha-non-natural amino acids and insert in the same protein.For example, in the presence of the sudden change O-tRNA with anticodon loop (for example having 8-10nt anticodon loop at least) (include, but is not limited to specific frameshit and suppress sub-tRNA), four or more polybase base codon pronounced single amino acid.In other embodiments, at least one four base codon of anticodon loop decodable code (including, but is not limited to), at least one five base codon or at least one hexabasic basic codon or more polybase base codon.Because there are 256 four possible base codons, so can use four or the polybase base codon multiple alpha-non-natural amino acid of in same cell, encoding more.Referring to people such as Anderson, (2002) Exploring the Limits of Codon and Anticodon Size,Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selectionof Efficient Suppressors of Four-base Codons and Identification of " Shifty " Four-baseCodons with a Library Approach in Escherichia coliJ.Mol.Biol.307:755-769.

For example, use the external biological synthetic method, four base codons have been used for incorporating alpha-non-natural amino acid into albumen.For example referring to people such as Ma, (1993)Biochemistry, 32:7939; With people such as Hohsaka, (1999)J.Am.Chem.Soc,121:34.CGGG and AGGU are used for incorporating the NBD derivative of 2-naphthyl L-Ala and Methionin into streptavidin that external frameshit with two chemical acylation suppresses sub-tRNA simultaneously.For example referring to people such as Hohsaka, (1999)J.Am.Chem.Soc, 121:12194.In in one, studying, people such as Moore check has the ability of tRNALeu derivative inhibition UAGN (N can be U, A, G or the C) codon of NCUA anticodon, and find that tetrad UAGA can be decoded with 13 to 26% efficient by the tRNALeu with UCUA anticodon, has seldom decoding in 0 or-1 frame.Referring to people such as Moore, (2000)J.Mol.Biol.,298:195.In one embodiment, can be used among the present invention based on the prolongation codon of rare codon or nonsense codon, its missense that can reduce other unwanted site is readed over frameshit and is suppressed.

For giving fixed system, select codon also can comprise a kind of in the natural three base codons, wherein the endogenous system does not use (or seldom using) natural three base codons.For example, it comprise the system of the tRNA that lacks the natural three base codons of identification and/or wherein three base codons be the system of rare codon.

Select codon to comprise the non-natural base pair according to circumstances.These non-natural base pairs further expand existing genetic alphabet table.Extra base pair is increased to 125 with the number of codeword triplet from 64.The characteristic of three base pairs comprises stable and optionally base pairing, with polysaccharase high frequency high fidelity ground effectively enzymatic incorporate among the DNA and effectively continuous primer extension after synthetic newborn non-natural base pair.For can the description through adjusting for example comprising people such as Hirao with the non-natural base pair that is used for method and composition, (2002) An unnatural base pair-for incorporating amino acidanalogues into protein,Nature BiotechnoloRy, 20:177-182.Other relevant open case is enumerated hereinafter.

For using in the body, non-natural nucleoside be membrane permeability and through phosphorylation to form corresponding triguaiacyl phosphate.In addition, the genetic information of increase is stable and is not destroyed by cellular enzymes.Benner and other people previous effort utilization are different from the right hydrogen bonding pattern of typical Watson-Crick, and its most noticeable example is that iso-C:iso-G is right.For example referring to people such as Switzer, (1989)J.Am.Chem.Soc, 111:8322; With people such as Piccirilli, (1990)Nature, 343:33; Kool, (2000)Curr.Opin.Chem.Biol, 4:602.Generally speaking, these bases are to a certain extent with natural base mispairing and can not be duplicated by enzymatic.Kool and co-worker thereof confirm that the hydrophobicity between the base loads interaction replaceable hydrogen bonding to drive the formation of base pair.Referring to Kool, (2000)Curt.Opin.Chem.Biol, 4:602; With Guckian and Kool, (1998)Angew.Chem.Int.Ed.Engl., 36,2825.Satisfy in the work of non-natural base pair of all above-mentioned requirements in exploitation, Schultz, Romesberg and co-worker thereof are systematically synthetic and study a series of non-natural hydrophobicity bases.Find that PICS:PICS self pairing is more more stable than natural base pair, and can incorporate among the DNA effectively by the Klenow fragment (KF) of e. coli dna polymerase I.For example referring to people such as McMinn, (1999)J.Am.Chem.Soc,121:11586; With people such as Ogawa, (2000)J.Am.Chem.Soc, 122:3274.Can KF synthetic 3MN:3MN self pairing under efficient enough for biological function and selectivity.For example referring to people such as Ogawa, (2000)J.Am.Chem.Soc, 122:8803.Yet two kinds of bases are all served as chain terminator and are further duplicated being used for.Developed recently and can be used for to duplicate PICS self paired mutant DNA polymerases.In addition, reproducible 7AI self pairing.For example referring to people such as Tae, (2001)J.Am.Chem.Soc, 123:7439.Also developed novel metal base pair Dipic:Py, it forms stable pairing in conjunction with Cu (II) time.Referring to people such as Meggers, (2000)J.Am.Chem.Soc, 122:10714.Because prolonging codon and non-natural codon is intrinsic orthogonal for natural codon, so method of the present invention can utilize this characteristic to produce quadrature tRNA from it.

The translation bypath system also can be used for alpha-non-natural amino acid incorporate into want in the polypeptide.In the translation bypath system, incorporate into big sequence in the gene rather than translate into albumen.Described sequence contains serves as instruction to induce rrna and skip described sequence and to continue the structure of the translation in downstream, insertion point.

In certain embodiments, by associated protein in nucleic acid encoding method of the present invention and/or the composition or polypeptide (or its part).Nucleic acid comprises at least one usually and selects codon, at least two selection codons, at least three selection codons, at least four selection codons, at least five selection codons, at least six selection codons, at least seven selection codons, at least eight selection codons, at least nine selection codons, selects codon more than ten or ten.

Can use the well-known and described in this article method of those skilled in the art to bring out the transgenation of coding associated protein or polypeptide, to comprise that for example one or more select codons to be used to incorporate into alpha-non-natural amino acid.For example, bring out the nucleic acid mutation that is used for associated protein and select codon, thereby incorporating into of one or more alpha-non-natural amino acids is provided to comprise one or more.The present invention includes any proteic any described anomaly (including, but is not limited to mutant) variant, for example comprise at least a alpha-non-natural amino acid.Similarly, the present invention also comprises corresponding nucleic acid, promptly has any nucleic acid of the selection codon of one or more one or more alpha-non-natural amino acids of encoding.

The nucleic acid molecule of coding such as the associated protein of hGH polypeptide can easily suddenly change and introduce halfcystine with any position of being wanted at polypeptide.Halfcystine is widely used in reactive molecule, water-soluble polymers, albumen or multiple other molecule is incorporated on the associated protein.Be applicable to that the method that halfcystine is introduced the position of wanting of hGH polypeptide is well-known in affiliated field, such as United States Patent (USP) the 6th, 608, technology is brought out in method described in No. 183 (it is incorporated herein by reference) and standard sudden change.

IV. non-naturally encoded amino acids

Multiple non-naturally encoded amino acids is applicable among the present invention.The non-naturally encoded amino acids of any number can be introduced in the hGH polypeptide.Generally speaking, the non-naturally encoded amino acids of being introduced is chemically inert to the amino acid (being L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) of 20 kinds of common genes encodings substantially.In certain embodiments, non-naturally encoded amino acids comprise with 20 kinds of common amino acids in undiscovered functional group (including, but is not limited to azido-, ketone group, aldehyde radical and aminooxy) effectively and optionally react to form the side chain functional group of stable engagement thing.For example, comprise the non-naturally encoded amino acids that contains the azido-functional group the hGH polypeptide can with polymkeric substance (including, but is not limited to gather (ethylene glycol)) or second polypeptide reaction that contains alkynyl moiety forming the stable engagement thing, it is with formation Huisgen[3+2 by nitrine and alkynes functional group selective reaction] the cycloaddition product produces.

The universal architecture of alpha amino acid is described as follows (formula I):

Non-naturally encoded amino acids is generally any structure (wherein the R base is any substituting group the used substituting group in 20 kinds of natural amino acids) with above-mentioned formula, and applicable to the present invention.Because non-naturally encoded amino acids of the present invention only is different from natural amino acid usually on side-chain structure, thus non-naturally encoded amino acids with the polypeptide of natural generation in form the identical mode of amido linkage and other amino acid (including, but is not limited to natural or non-naturally encoded amino acids) formation amido linkage.Yet non-naturally encoded amino acids has distinguishes the side-chain radical that comes with itself and natural amino acid.For example, R comprise according to circumstances alkyl-, aryl-, acyl group-, ketone group-, azido--, hydroxyl-, hydrazine, cyano group-, halogen-, hydrazides, thiazolinyl, alkynyl, ether, mercaptan, seleno-, alkylsulfonyl-, borate,

Acid group, phosphate, phosphonate group, phosphine, heterocycle, ketenes, imines, aldehyde, ester, thioic acid sulfoacid, azanol, amino or the like or its any combination.Other applicable to relevant non-natural of the present invention produce amino acid include, but is not limited to comprise can photic activatory linking agent amino acid, the amino acid of spin labeling, fluorescence amino acid, the melts combine acidic amino acid, containing metal amino acid, radioactivity amino acid, amino acid with novel functional group, covalently or non-covalently with the amino acid of other interaction of molecules, but the amino acid of light sealing and/or photoisomerization, the amino acid that comprises vitamin H or vitamin H analogue, glycosylation amino acid (such as through sugar-substituted Serine), amino acid through other carbohydrate modification, ketone group containing amino acid, the amino acid that comprises polyoxyethylene glycol or polyethers, amino acid through the heavy atom replacement, but but the amino acid of chemical cracking and/or photo-cleavage, compare with natural amino acid and to have the prolongation side chain (include, but is not limited to polyethers or long chain hydrocarbon, include, but is not limited to greater than about 5 or greater than about 10 carbon) amino acid, what be connected with carbon contains glycoprotein amino acid, redox active amino acids, contain the amino acid of amino thioic acid sulfoacid and comprise one or more toxicity amino acid partly.

Applicable to the present invention and be applicable to exemplary non-naturally encoded amino acids with water-soluble polymers reaction include, but is not limited to those have carbonyl, aminooxy, hydrazine, hydrazides, Urea,amino-, the amino acid of azido-and alkyne reaction base.In certain embodiments, non-naturally encoded amino acids comprises sugar moieties.Described amino acid whose example comprises N-ethanoyl-L-glucose amido-L-Serine, N-ethanoyl-L-semi-lactosi amido-L-Serine, N-ethanoyl-L-glucose amido-L-Threonine, N-ethanoyl-L-glucose amido-altheine and O-seminose amido-L-Serine.Described amino acid whose example comprises also wherein between the amino acid and sugar that naturally occurring N-or O-bonding are by at the common undiscovered covalent linkage of occurring in nature (including, but is not limited to alkene, oxime, thioether, acid amides or the like) metathetical example.Described amino acid whose example is also included within common undiscovered sugar in the naturally occurring albumen, such as 2-deoxidation-glucose, 2-deoxy-galactose or the like.

The many non-naturally encoded amino acids that provided herein can available from for example Sigma-Aldrich (St.Louis, MO, USA), Novabiochem (EMD Biosciences, Darmstadt, the branch office of Germany) or Peptech (Burlington, MA, USA).The amino acid that can not buy provides by this paper according to circumstances and synthesizes or use the known standard method of those skilled in the art to synthesize.About organic synthesis technology for example referring to Fessendon and FessendonOrganic Chemistry(1982, the 2 editions, Willard Grant Press, Boston Mass.); March'sAdvanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and SundbergAdvanced Organic Chemistry(the 3rd edition, part A and B, 1990, Plenum Press, NewYork).Also referring to U.S. Patent Application Publication case 2003/0082575 and 2003/0108885, it is incorporated herein by reference.Except that the alpha-non-natural amino acid that contains novel side chain, also comprise modified skeleton structure according to circumstances applicable to alpha-non-natural amino acid of the present invention, it includes, but is not limited to by the illustrated structure of the structure of formula II and III:

Wherein Z comprises OH, NH usually₂, SH, NH-R ' or S-R '; X and Y can be identical or different, and it comprises S or O usually; And R and R ' are identical or different according to circumstances, and it normally is selected from the same composition tabulation and the hydrogen of the above-mentioned R group of the alpha-non-natural amino acid that is used to have formula I.For example, alpha-non-natural amino acid of the present invention comprises replacement according to circumstances in amino or carboxyl, and is illustrated suc as formula II and III.Such alpha-non-natural amino acid includes, but is not limited to alpha hydroxy acid, α-thioic acid sulfoacid, alpha-amino group carbothioic acid ester, includes, but is not limited to have side chain or non-natural side chain corresponding to common 20 kinds of natural amino acids.In addition, the replacement on alpha-carbon includes, but is not limited to L, D or α-α-disubstituted amino acid according to circumstances, such as D-glutamate, D-L-Ala, D-methyl-O-tyrosine, aminobutyric acid or the like.Other analog comprises cyclic amino acid (such as proline analogs and 3,4,6,7,8 and 9 yuan of loop proline analogues) and β and γ amino acid (such as being substituted Beta-alanine and γ-An Jidingsuan).

Many alpha-non-natural amino acids are based on the natural amino acid such as tyrosine, glutamine, phenylalanine or the like, and are applicable among the present invention.The tyrosine analogue include, but is not limited to the tyrosine through para-orientation, the tyrosine that replaces through the ortho position and through between the tyrosine that replaces of position, wherein be substituted tyrosine and comprise (including, but is not limited to) ketone group (including, but is not limited to ethanoyl), benzoyl, amino, hydrazine, azanol, thiol group, carboxyl, sec.-propyl, methyl, C₆-C₂₀Straight or branched hydrocarbon, saturated or unsaturated hydrocarbons, O-methyl, polyether-based, nitro, alkynyl or the like.In addition, also contain through polysubstituted aryl rings.The glutamine derivative that includes, but is not limited to Alpha-hydroxy derivative, γ substitutive derivative, cyclic derivatives and replace applicable to glutamine analogue of the present invention through acid amides.Applicable to the example of phenylalanine analogues of the present invention include, but is not limited to the phenylalanine through para-orientation, the phenylalanine that replaces through the ortho position and through between the phenylalanine that replaces of position, wherein substituting group comprises (including, but is not limited to) hydroxyl, methoxyl group, methyl, allyl group, aldehyde, azido-, iodine, bromine, ketone group (including, but is not limited to ethanoyl), benzoyl, alkynyl or the like.Particular instance applicable to alpha-non-natural amino acid of the present invention includes, but is not limited to ethanoyl-L-phenylalanine; O-methyl-L-tyrosine; L-3-(2-naphthyl) L-Ala; the 3-methylphenylalanine; O-4-allyl group-L-tyrosine; 4-propyl group-L-tyrosine; three-O-ethanoyl-GlcNAc β-Serine; L-Dopa; fluoridize phenylalanine; sec.-propyl-L-phenylalanine; to azido--L-phenylalanine; to acyl group-L-phenylalanine; to benzoyl-L-phenylalanine; L-phosphate Serine; the phosphonate group Serine; phosphonate group tyrosine; to the phenyl-iodide L-Ala; to the bromobenzene L-Ala; to amino-L-phenylalanine; sec.-propyl-L-phenylalanine and to alkynes propoxy--phenylalanine or the like.Example applicable to the structure of multiple alpha-non-natural amino acid of the present invention for example is provided in to be entitled as among the WO 2002/085923 of " In vivo incorporation of unnatural aminoacids ".About extra methionine(Met) analogue, also referring to people such as Kiick, (2002) Incorporation of azides into recombinant proteins for chemoselective modification by theStaudinger ligation,PNAS99:19-24.

The composition of the hGH polypeptide that comprises alpha-non-natural amino acid (such as to alkynes propoxy--phenylalanine) is provided in one embodiment.The various compositions that comprise alkynes propoxy--phenylalanine and (including, but is not limited to) albumen and/or cell also are provided.On the one hand, comprise that the composition to alkynes propoxy--phenylalanine alpha-non-natural amino acid further comprises quadrature tRNA.But alpha-non-natural amino acid bond (including, but is not limited to covalently) is to quadrature tRNA, include, but is not limited to via amino-acyl bond covalently bond to quadrature tRNA, covalently bond is to 3 ' OH of the terminal ribose of quadrature tRNA or 2 ' OH etc.

Can incorporate that chemical part in the albumen provides various advantages into and to proteic manipulation via alpha-non-natural amino acid.For example, ketone group functional group unique reactive allows with any selective modification albumen in vitro and in vivo in the multiple reagent that contains hydrazine or azanol.For example, the heavy atom alpha-non-natural amino acid can be used for phasing x-ray structure data.Use alpha-non-natural amino acid locus specificity ground to introduce heavy atom also can be used for providing selectivity and handiness aspect the position of heavy atom in selection.For example, photoreactivity alpha-non-natural amino acid (including, but is not limited to have the amino acid of benzophenone and aromatic yl azide (including, but is not limited to azidomethyl phenyl nitrogenize thing) side chain) allows in the body and the external albumen of photocrosslinking effectively.The example of photoreactivity alpha-non-natural amino acid includes, but is not limited to azido--phenylalanine with to benzoyl-phenylalanine.Can come at random crosslinked albumen by exciting the of short duration control that the photoresponse base is provided then with photoreactivity alpha-non-natural amino acid.In one example, the methyl of alpha-non-natural amino acid can be by through isotope-labeled (including, but is not limited to) methyl substituted, to use nucleus magnetic resonance and vibrational spectrum class hour as local structure and dynamic (dynamical) probe (including, but is not limited to).For example, alkynyl or azido-functional group allow via [3+2] cycloaddition reaction with the molecular selectivity modified protein.

The alpha-non-natural amino acid of incorporating in the polypeptide at N-terminal can and be different from the NH that exists usually by the R group in a-amino acid (referring to formula I)₂Second reactive group of group is formed, and described R group is any substituting group the used substituting group in 20 kinds of natural amino acids.Can incorporate similar alpha-non-natural amino acid at C-terminal with second reactive group that is different from the COOH group that exists usually in the a-amino acid (referring to formula I).

The chemosynthesis of alpha-non-natural amino acid

Be applicable to many alpha-non-natural amino acids of the present invention for example can available from Sigma (USA) or Aldrich (Milwaukee, WI, USA).The amino acid that can not buy provides according to this paper according to circumstances or provides according to institute in the various open cases and synthesizes or use the known standard method of those skilled in the art to synthesize.About organic synthesis technology for example referring to Fessendon and FessendonOrganic Chemistry(1982, the 2 editions, Willard Grant Press, BostonMass.); March'sAdvanced Organic Chemistry(the 3rd edition, 1985, Wiley and Sons, New York); With Carey and SundbergAdvanced Organic Chemistry(the 3rd edition, part A and B, 1990, PlenumPress, New York).Description alpha-non-natural amino acid synthetic additionally discloses case and for example comprises the WO 2002/085923 that is entitled as " In vivoincorporation of Unnatural Amino Acids "; People such as Matsoukas, (1995)J.Med.Chem.,38,4660-4669; King, F.E. ﹠amp; Kidd, D.A.A. (1949) A New Synthesisof Glutamine and of γ-Dipeptides of Glutamic Acid from Phthylated Intermediates.J.Chem.Soc,3315-3319; Friedman, O.M. ﹠amp; Chatterrji, R. (1959) Synthesis of Derivatives ofGlutamine as Model Substrates for Anti-Tumor Agents.J.Am.Chem.Soc.81,3750-3752; Craig, people such as J.C., (1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl] amino] quinoline (Chloroquine).J.Ore.Chem.53,1167-1170; Azoulay, M., Vilmont, M. ﹠amp; Frappier, F. (1991) Glutamine analogues asPotential Antimalarials.Eur.J.Med.Chem.26,201-5; Koskinen, A.M.P. ﹠amp; Rapoport, H. (1989) 5ynthesis of 4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues.J.Org.Chem.54,1859-1866; Christie, B.D. ﹠amp; Rapoport, H. (1985) Synthesis ofOptically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through Amino Acid Decarbonylation and Iminium Ion Cyclization.J.Org.Chem.1989:1859-1866; People such as Barton, (1987) Synthesis of Novel a-Amino-Acids andDerivatives Using Radical Chemistry:Synthesis of L-and D-a-Amino-Adipic Acids, L-a-aminopimelic Acid and Appropriate Unsaturated Derivatives.Tetrahedron Lett.43:4297-4308; With people such as Subasinghe, (1992) Quisqualic acid analogues:synthesis ofbeta-heterocyclic 2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site.J.Med.Chem.35:4602-7.Also referring to No. the 10/744th, 899, the patent application case that is entitled as " Protein Arrays " of application on December 22nd, 2003 and the 60/435th, No. 821 of application on December 22nd, 2002.

A. carbonyl reaction base

Amino acid with carbonyl reaction base allows especially to come via nucleophilic addition(Adn) or aldol reaction the various reactions of link molecule (including, but is not limited to PEG or other water soluble molecules).

The exemplary carbonylamino acid that contains can be as follows:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R₃For H, amino acid, polypeptide or N-terminal are modified base, and R₄For H, amino acid, polypeptide or C-terminal are modified base.In certain embodiments, n is 1, R₁Be phenyl and R₂For simple alkyl (being methyl, ethyl or propyl group) and ketone partly are arranged in contraposition with respect to alkyl group side chain.In certain embodiments, n is 1, R₁Be phenyl and R₂For simply alkyl (being methyl, ethyl or propyl group) and ketone partly are arranged in respect to position between alkyl group side chain.

Ethanoyl-(+/-)-phenylalanine and an ethanoyl-(+/-)-phenylalanine synthetic is described in people such as Zhang, Z., and among the Biochemistry 42:6735-6746 (2003), it is incorporated herein by reference.Other contains carbonylamino acid and can be prepared similarly by the those skilled in the art.

In certain embodiments, the polypeptide that comprises non-naturally encoded amino acids through chemically modified to produce reactive carbonyl functional group.For example, be applicable to that the aldehyde functional group who engages reaction can produce by having contiguous functional group amino and hydroxyl.When bioactive molecules was polypeptide, for example N-terminal Serine or Threonine (it can exist usually or can expose via chemistry or enzymatic digestion) were used in the mild oxidation cracking condition that uses periodate and produce the aldehyde functional group down.For example referring to people such as Gaertner, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K. ﹠amp; Stroh, L, Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J.Biol.Chem.269:7224-7230 (1994).Yet known method is confined to the amino acid of peptide or proteic N-terminal in the affiliated field.

In the present invention, having contiguous hydroxyl and amino non-naturally encoded amino acids can be used as " through camouflage " aldehyde functional group and incorporates in the polypeptide.For example, the 5-oxylysine has the hydroxyl that is adjacent to ε amine.The reaction conditions that is used to produce aldehyde be usually included in add molar excess under the mild conditions sodium periodate to avoid other site oxidation in polypeptide.The pH value of oxidizing reaction is generally about 7.0.Type reaction comprises that the sodium periodate with about 1.5 molar excess adds in the buffered polypeptide solution, cultivates about 10 minutes subsequently in the dark.For example referring to United States Patent (USP) the 6th, 423, No. 685, it is incorporated herein by reference.

The carbonyl functional group can with contain hydrazine, hydrazides, azanol or Urea,amino-reagent selective reaction in the aqueous solution under mild conditions, to form corresponding hydrazone, oxime or semicarbazone bonding respectively, it is stable under physiological condition.For example referring to Jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); Shao, J. and Tarn, J.P., J.Am.Chem.Soc.117:3893-3899 (1995).In addition, selective modification is carried out in the unique reactive permission of carbonyl in the presence of other amino acid side chain.For example referring to Cornish, people such as V.W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F. ﹠amp; Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L.K., Science 276:1125-1128 (1997).

B. hydrazine, hydrazides or Urea,amino-reactive group

Contain such as hydrazine, hydrazides or Urea,amino-the non-naturally encoded amino acids of nucleophilic group allow to react to form joiner (include, but is not limited to and PEG or other water-soluble polymers form joiner) with various electrophilic groups.

Exemplary contain hydrazine, hydrazides or Urea,amino-amino acid can be as follows:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.

In certain embodiments, n is 4, R₁Do not exist and X is N.In certain embodiments, n is 2, R₁Do not exist and X does not exist.In certain embodiments, n is 1, R₁Be phenyl, X is O, and Sauerstoffatom is positioned at the contraposition of aliphatic group on the aryl rings.

Contain hydrazides, hydrazine and Urea,amino-amino acid can buy from commercial source.For example, L-L-glutamic acid-γ-hydrazides can (St.Louis MO) buys from Sigma Chemical.Other amino acid that can not buy can be prepared by the those skilled in the art.For example referring to United States Patent (USP) the 6th, 281, No. 211, it is incorporated herein by reference.

Contain have hydrazides, the polypeptide of hydrazine or Urea,amino-functional group's non-naturally encoded amino acids can reach optionally reaction effectively with the various molecules that contain aldehyde or have similar chemically reactive other functional group.For example referring to Shao, J. and Tam, J.Am.Chem.Soc.117:3893-3899 (1995).Unique reactivity of hydrazides, hydrazine and Urea,amino-functional group makes it compare with the nucleophilic group (including, but is not limited to hydroxyl or lysine amino and N-terminal on Serine or the Threonine) that exists on 20 kinds of common amino acids, has more reactivity significantly for aldehyde, ketone and other electrophilic group.

C. the amino acid that contains aminooxy

The non-naturally encoded amino acids that contains aminooxy (being also referred to as azanol) allows to react to form joiner (including, but is not limited to form joiner with PEG or other water-soluble polymers) with various electrophilic groups.Be similar to hydrazine, hydrazides and Urea,amino-, aminooxy enhanced nucleophilicity allows itself and the various molecule that contains aldehyde or have similar chemically reactive other functional group to reach optionally reaction effectively.For example referring to Shao, J. and Tam, J., J.Am.Chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, Ace.Chem.Res.34:727-736 (2001).Though with the reaction result of diazanyl is corresponding hydrazone, yet, oxime obtained usually by aminooxy and reaction such as the carbonyl group-containing groups of ketone.

The exemplary amino acid that contains aminooxy can be as follows:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; Y=C (O) or do not exist; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.In certain embodiments, n is 1, R₁Be phenyl, X is O, and m is 1 and has a Y.In certain embodiments, n is 2, R₁Do not exist with X, m be 0 and Y do not exist.

The amino acid that contains aminooxy can be by amino acid presoma (homoserine, Serine and the Threonine) preparation of easy acquisition.For example referring to M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858 (2003).From natural origin separate such as L-2-amino-4-(aminooxy) butyro-some contain the amino acid (Rosenthal, people such as G., Life Sci.60:1635-1641 (1997)) of aminooxy.Other amino acid that contains aminooxy can be prepared by the those skilled in the art.

D. nitrine and alkyne reaction base

Unique reactivity of nitrine and alkynes functional group makes it very be applicable to selective modification polypeptide and other biomolecules.Organic azide (especially aliphatic trinitride) and alkynes are normally stable for reactive electrochemical conditions commonly used.Specific, nitrine and alkynes functional group are inert for the side chain (being the R group) of 20 kinds of common amino acids finding in the polypeptide of natural generation.Yet, when azido-and alkynyl closely near the time, its " springload " character disclosed and its via Huisgen[3+2] cycloaddition reaction reaches effectively and optionally reacts to produce corresponding triazole.For example referring to people such as ChinJ., Science 301:964-7 (2003); Wang, people such as Q., J.Am.Chem.Soc.125,3192-3193 (2003); Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction comprise the selectivity cycloaddition reaction (for example referring to Padwa, A., COMPREHENSIVE ORGANIC SYNTHESIS, the 4th volume, (Trost, B.M. compile, 1991), 1069-1109 page or leaf; Huisgen, R.1,3-DiPOLAR CYCLOADDITION CHEMISTRY, (Padwa, A. compile, 1984), 1-176 page or leaf) rather than nucleophilic substitution, thus incorporate into the non-naturally encoded amino acids with the side chain that contains nitrine and alkynes can allow the gained polypeptide on the position of non-naturally encoded amino acids through selective modification.(include, but is not limited to the CuSO of catalytic amount by the Cu (II) that in the presence of the reductive agent that is used for Cu (II) is reduced into Cu (I) then and there, adds catalytic amount₄The cycloaddition reaction that form), can at room temperature under aqueous conditions, relate to the hGH polypeptide that contains nitrine or alkynes.For example referring to Wang, people such as Q., J.Am.Chem.Soc.125,3192-3193 (2003); Tornoe, people such as C.W., J.Org.Chem.67:3057-3064 (2002); People such as Rostovtsev, Angew.Chem.Iht.Ed.41:2596-2599 (2002).Exemplary reductive agent includes, but is not limited to ascorbate salt, metallic copper, quinine, quinhydrones, vitamin K, gsh, halfcystine, Fe²⁺, Co²⁺With the electromotive force that applies.

At the Huisgen[3+2 that needs between nitrine and the alkynes] under the certain situation of cycloaddition reaction, the hGH polypeptide comprises the non-naturally encoded amino acids of alkynyl moiety and is interconnected in amino acid whose water-soluble polymers and comprises the nitrine part.Perhaps, also can carry out reversed reaction (promptly with amino acid on nitrine part and water-soluble polymers on the alkynyl moiety reversed reaction that exists).

The nitrine functional group also can with contain aryl ester and suitably optionally react to produce amido linkage through the functionalized water-soluble polymers of aryl phosphine part.The aryl phosphine group reduces trinitride then and there, and gained amine reacts to produce corresponding acid amides effectively with contiguous ester linkage then.For example referring to E.Saxon and C.Bertozzi, Science 287,2007-2010 (2000).The amino acid that contains nitrine can be alkyl azide (including, but is not limited to 2-amino-6-azido--1-caproic acid) or aromatic yl azide (to azido--phenylalanine).

The exemplary water-soluble polymers that contains aryl ester and phosphine part can be as follows:

Wherein X can be O, N, S or does not exist, and Ph is a phenyl, and W is that water-soluble polymers and R can be H, alkyl, aryl, are substituted alkyl and are substituted aryl.Exemplary R group includes, but is not limited to-CH₂,-C (CH₃)₃,-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O)₂R ' ,-S (O)₂NR ' R " ,-CN and-NO₂R ', R ", R  and R " " the assorted alkyl that means hydrogen independently of one another, is substituted or is unsubstituted, the aryl that is substituted or is unsubstituted (include, but is not limited to replace aryl), alkyl, alkoxyl group or the thioalkoxy group or the arylalkyl that are substituted or are unsubstituted through 1-3 halogen.When compound of the present invention comprised an above R group, for example, each R group all was independent selection, when having R ', R ", R  and R " " when one of group is above, each R ', R ", R  and R " " the group situation is identical.As R ' and R " when being connected in identical nitrogen-atoms, it can make up to form 5,6 or 7 yuan of rings with nitrogen-atoms.For example ,-and NR ' R " mean and include, but is not limited to 1-pyrrolidyl and 4-morpholinyl.According to substituent above the discussion, be understood by those skilled in the art that term " alkyl " means and comprise following group: it comprises the carbon atom that is attached to the group outside the dehydrogenation base, (includes, but is not limited to-CF such as haloalkyl₃With-CH₂CF₃) and acyl group (include, but is not limited to-C (O) CH₃,-C (O) CF₃,-C (O) CH₂OCH₃Or the like).

The nitrine functional group also can with contain thioesters and suitably optionally react to produce amido linkage through the functionalized water-soluble polymers of aryl phosphine part.The aryl phosphine group reduces trinitride then and there, and gained amine reacts to produce corresponding acid amides effectively with the thioesters bonding then.The exemplary water-soluble polymers that contains thioesters and phosphine part can be as follows:

Wherein n is 1-10; X can be O, N, S or does not exist, and Ph is a phenyl, and W is a water-soluble polymers.

The exemplary alkynyl amino acid that contains can be as follows:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.In certain embodiments, n is 1, R₁Be phenyl, X does not exist, m be 0 and acetylene moiety be arranged in contraposition with respect to alkyl group side chain.In certain embodiments, n is 1, R₁Be phenyl, X is O, m be 1 and the alkynes propoxy-be arranged in contraposition (being O-propargyl-tyrosine) with respect to alkyl group side chain.In certain embodiments, n is 1, R₁Do not exist with X and m is 0 (being PGIY).

Containing alkynyl amino acid can be buied by the market.For example, PGIY can (Burlington MA) buys by Peptech.Perhaps, containing alkynyl amino acid can come according to the standard method preparation.For example, can be as (for example) Deiters, A. wait the people, come synthetic described in the J.Am.Chem.Soc.125:11782-11783 (2003) to alkynes propoxy-phenylalanine, and as Kayser, B. wait the people, Tetrahedron 53 (7): the described 4-alkynyl-L-phenylalanine that synthesizes of 2475-2484 (1997).Other contains alkynyl amino acid and can be prepared by the those skilled in the art.

The exemplary nitrine amino acid that contains can be as follows:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base, and R₃For H, amino acid, polypeptide or C-terminal are modified base.In certain embodiments, n is 1, R₁Be phenyl, X does not exist, m be 0 and nitrine partly be arranged in contraposition with respect to alkyl group side chain.In certain embodiments, n is 0-4 and R₁Do not exist with X, and m=0.In certain embodiments, n is 1, R₁Be phenyl, X is O, m be 2 and β azido-oxyethyl group partly be arranged in contraposition with respect to alkyl group side chain.

Containing nitrine amino acid can buy from the market.For example, 4-triazobenzene L-Ala can be from Chem-ImpexInternational, and (Wood Dale IL) obtains Inc..For containing for the nitrine amino acid that those can not be buied; can use the known standard technique of those skilled in the art relatively easily to prepare azido-, include, but is not limited to via the suitable leaving group (including, but is not limited to halogenide, methanesulfonate, tosylate) of displacement or via the lactone of open loop through suitable protection.For example referring to March'sAdvanced Organic Chemistry(the 3rd edition, 1985, Wiley andSons, New York).

E. amineothiot reactive group

Unique reactivity of the amineothiot functional group who replaces through β makes it very be applicable to via forming thiazolidine and optionally modifies polypeptide and other biomolecules that contains aldehyde radical.For example referring to J.Shao and J.Tam, J.Am.Chem.Soc.1995,117 (14) 3893-3899.In certain embodiments, the amineothiot amino acid that replaces through β can be incorporated in the hGH polypeptide and react with the water-soluble polymers that comprises the aldehyde functional group then.In certain embodiments, water-soluble polymers, medicine joiner or other useful load can be via forming thiazolidine with comprise the amino acid whose hGH polypeptide of the amineothiot coupling that replaces through β.

The cellular uptake of alpha-non-natural amino acid

When design and when selecting to be used for (including, but is not limited to) and incorporating the alpha-non-natural amino acid of albumen into, eukaryotic cell is a common problem of considering to the picked-up of alpha-non-natural amino acid.For example, the high charge density of a-amino acid shows that these compounds unlikely have cell permeability.Natural amino acid via a series of based on proteic delivery system and being shot being taken in the eukaryotic cell.Can evaluate which kind of alpha-non-natural amino acid (if having) by the rapid screening of cellular uptake.For example referring to No. the 10/744th, 899, the application case that is entitled as " Protein Arrays " of application on December 22nd, 2003 and the 60/435th, No. 821 and Liu of application on December 22nd, 2002, D.R.﹠amp; Schultz, P.G. (1999) Progresstoward the evolution of an organism with an expanded genetic code.PNAS United StatesToxicological detection among the 96:4780-4785.Though be easy to analyze picked-up with various calibratings, the alternative method that designs the alpha-non-natural amino acid that is subjected to the effect of cellular uptake path is for providing the biosynthesizing path to produce amino acid in the body.

The biosynthesizing of alpha-non-natural amino acid

Existed many biosynthesizing path to be used to produce amino acid and other compound in the cell.Though may not have the biosynthetic means that is used for specific alpha-non-natural amino acid at occurring in nature (including, but is not limited in eukaryote), the invention provides described method.For example, by adding novel enzyme or modifying existing host cell path, in host cell, produce the biosynthesizing path that is used for alpha-non-natural amino acid according to circumstances.Extra novel enzyme is the enzyme or the artificial enzyme that develops of natural generation according to circumstances.For example, the biosynthesizing of p-Aminophenylalanine (for example presenting in the WO 2002/085923 that is entitled as " In vivoincorporation of unnatural amino acids ") depends on the adding from the combination of other organic known enzyme.Can be by described gene being introduced eukaryotic cell with the plasmid transformant that comprises the gene that is used for these enzymes.Described gene provides the enzymatic path with the synthetic compound of being wanted when expressing in cell.The example of the enzyme type that adds according to circumstances is provided in following example.Extra enzyme sequence for example is found among the Genbank.The enzyme that also will manually develop in the same manner according to circumstances adds in the cell.The cell mechanism of manipulated cell and resource are to produce alpha-non-natural amino acid by this way.

The whole bag of tricks can be used for producing novel enzymes to be used for the biosynthesizing path or to be used to the existing route that develops.For example, use recurrence reorganization (including, but is not limited to by Maxygen the recurrence reorganization of Inc. (can on World Wide Web maxygen.com, obtain) exploitation) to develop novel enzymes and path according to circumstances.For example referring to Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling,Nature370 (4): 389-391; And Stemmer, (1994), DNA shuffling by random fragmentation and reassembly:In vitro recombination formolecular evolution,Proc.Natl.Acad.Sci.USA., 91:10747-10751.Similarly, the DesignPath that develops by Genencor (can on World Wide Web genencor.com, obtain)^TMBe used for the metabolic pathway engineering design according to circumstances, (including, but is not limited to) is used for producing O-methyl-L-tyrosine at cell with the engineering design path.This technology uses (including, but is not limited to) to rebuild existing route via the combination of the novel gene of functional genomics identification and molecular evolution and design in host's organism.Diversa company (can obtain on World Wide Web diversa.com) also is provided for the technology (including, but is not limited to) in library in rapid screening gene and gene path to produce novel path.

Concentration with the alpha-non-natural amino acid that produces through the biosynthesizing path of engineering design of the present invention should be enough to be used in effective protein proteins biosynthesizing (including, but is not limited to the n cell amount) usually, but can not reach the degree that influences other amino acid whose concentration or exhaust the cell resource.The typical concentration that produces in vivo arrives about 0.05mM for about 10mM by this way.With the plasmid transformant that comprises the gene that is used to produce the required enzyme of particular path and after producing alpha-non-natural amino acid, use the generation of selecting to be used for the alpha-non-natural amino acid of the synthetic and cell growth of ribosomal protein in the body according to circumstances with further optimization.

Polypeptide with alpha-non-natural amino acid

Can carry out incorporating into of alpha-non-natural amino acid for multiple purpose, described purpose comprises the change on (but being not limited to) customization protein structure and/or the function; Change the accessibility of size, acidity, nucleophilicity, hydrogen bond knot, hydrophobicity, proteolytic enzyme target site; Alignment portion (including, but is not limited to be used for protein arrays) etc.Can have enhancing new catalysis or biophysical properties fully even comprise the albumen of alpha-non-natural amino acid.For example, modify following characteristic in the albumen according to circumstances by alpha-non-natural amino acid is included: toxicity, bio distribution, structural performance, dichroism, chemistry and/or photochemical properties, catalytic capability, transformation period (including, but is not limited to serum half-life), with ability (including, but is not limited to covalently or non-covalently) of other molecular reaction or the like.Comprise that the proteic composition that comprises at least a alpha-non-natural amino acid is applicable to the research of (including, but is not limited to) novel therapeutic agents, diagnostic reagent, catalytic enzyme, industrial enzyme, conjugated protein (including, but is not limited to antibody) and (including, but is not limited to) protein structure and function.For example referring to Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function,Current OpinionIn Chemical Biology, 4:645-652.

In one aspect of the invention, composition comprises at least a albumen with at least one (including, but is not limited at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or more) alpha-non-natural amino acid.Alpha-non-natural amino acid can be identical or different, includes, but is not limited to exist in 1,2,3,4,5,6,7,8,9,10 or more different loci comprising 1,2,3,4,5,6,7,8,9,10 or how different alpha-non-natural amino acid in the albumen.On the other hand, composition comprises albumen, and wherein at least a (but being less than all) of existing specific amino acids replaces through alpha-non-natural amino acid in the albumen.For given albumen with more than one alpha-non-natural amino acids, alpha-non-natural amino acid can identical or different (include, but is not limited to the alpha-non-natural amino acid that albumen can comprise that two or more are dissimilar, perhaps can comprise two kinds of identical alpha-non-natural amino acids).For the given albumen with two or more alpha-non-natural amino acids, alpha-non-natural amino acid can be identical, different or be the combination of alpha-non-natural amino acid with at least a different alpha-non-natural amino acid of multiple identical type.

Associated protein or polypeptide with at least a alpha-non-natural amino acid are feature of the present invention.The present invention also comprises polypeptide with at least a alpha-non-natural amino acid or the albumen that uses the compositions and methods of the invention to produce.Also can there be vehicle (including, but is not limited to pharmaceutically acceptable vehicle) in the albumen.

By produce associated protein or the polypeptide with at least a alpha-non-natural amino acid in eukaryotic cell, albumen or polypeptide will be modified after will generally including eukaryotic translation.In certain embodiments, albumen comprises at least one alpha-non-natural amino acid and at least one by the posttranslational modification of carrying out in the eukaryotic cell body, and wherein said posttranslational modification be can't help prokaryotic cell prokaryocyte and carried out.For example, posttranslational modification include, but is not limited to that acetylize, acidylate, lipid-modified, palmitoylation, palmitinic acid addition, phosphorylation, glycolipid bonding are modified, glycosylation or the like.On the one hand, posttranslational modification comprises via GlcNAc-l-asparagine key oligosaccharides (is included, but is not limited to (GlcNAc-Man)₂-Man-GlcNAc-GlcNAc)) be connected in l-asparagine.Referring to table 1, it enumerates some examples (can have extra residue, it is not showed yet) of the oligosaccharides that is connected with N of eukaryotic protein.On the other hand, posttranslational modification comprises via GalNAc-Serine or GalNAc-Threonine bonding or GlcNAc-Serine or GlcNAc-Threonine bonding oligosaccharides (including, but is not limited to Gal-GalNAc, Gal-GlcNAc etc.) is connected in Serine or Threonine.

Table 1: via the example of the oligosaccharides of GlcNAc-bonding

On the other hand, posttranslational modification comprise presoma (including, but is not limited to thyrocalcitonin presoma, calcitonin-gene-related peptide presoma, Pre Pro PTH, preproinsulin, proinsulin, pre-pro-opiomelanocortin, proopiomelanocortin or the like) proteolysis processing, be assembled into many subunits albumen or macromole assembly, another site of translating in the cell (includes, but is not limited to organoid, such as endoplasmic reticulum, golgi body, nuclear, lysosome, peroxysome, plastosome, chloroplast(id), vacuole etc., perhaps via the secretion path).In certain embodiments, described albumen comprises that secretion or positioning sequence, epitope label, FLAG label, poly are histidine-tagged, GST merges or the like.United States Patent (USP) the 4th, 963,495 and 6,436, No. 674 (it is incorporated herein by reference) described in detail through design and constructed body with improvement hGH polypeptide excretory.

An advantage of alpha-non-natural amino acid is that it presents the extra chemical part that can be used for adding additional molecules.These modifications can form or external formation in the body in eucaryon or non-eukaryotic cell.Therefore, in certain embodiments, posttranslational modification is to pass through alpha-non-natural amino acid.For example, described posttranslational modification can be passed through nucleophilic-electrophilic reaction.The current proteic most covalent linkage that are included between nucleophilic and the electrophilic reaction collocation thing that react of selective modification that are used for form, and include, but is not limited to the reaction of α halogenated ketone and Histidine or cysteine side chain.Selectivity in these cases is by the quantity of nucleophilic residues in the albumen and accessibility decision.In albumen of the present invention, can use other to have more optionally reaction, such as non-natural ketone group amino acid and hydrazides or aminooxy compound in external or intravital reaction.For example, wait the people, (1996) referring to CornishAm.Chem.Soc, 118:8150-8151; Mahal waits the people, (1997)Science.276:1125-1128; Wang waits the people, (2001)Science292:498-500; Chin waits the people, (2002)Am.Chem.Soc.124:9026-9027; Chin waits the people, (2002)Proc.Natl.Acad.Sci., 99:11020-11024; Wang waits the people, (2003)Proc.Natl.Acad.Sci, 100:56-61; Zhang waits the people, (2003)Biochemistry, 42:6735-6746 and Chin wait the people, (2003)Science, compiling.It allows the almost any albumen of main selectivity mark with the reagent that comprises fluorophore, linking agent, sugar derivatives and cytotoxicity molecule.Also referring to No. the 10/686th, 944, U.S. patent application case, title is " Glycoprotein synthesis ", application on January 16th, 2003, and it is incorporated herein by reference.(including, but is not limited to) also can carry out (including, but is not limited to triaryl phosphine reagent) by the Staudinger connection via the amino acid whose posttranslational modification of azido-.For example referring to people such as Kiick, (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudinger ligation,PNAS99:19-24.

The invention provides another kind and be used for the proteic high efficiency method of selective modification, it comprises that the alpha-non-natural amino acid heredity that will (include, but is not limited to) to contain azido-or alkynyl part is incorporated into and replys in the albumen of selecting codon.These amino acid side chains then can be respectively with (including, but is not limited to) alkynyl or azido derivant by (including, but is not limited to) Huisgen[3+2] cycloaddition reaction modify (for example referring to Padwa, A.Comprehensive OrRanic Synthesis, the 4thVolume, compile (1991), Trost, B.M., Pergamon, Oxford, 1069-1109 page or leaf and Huisgen, R.1,3-DipolarCycloaddition Chemistry, compile (1984), Padwa, A., Wiley, New York, 1-176 page or leaf).Because this method comprises cycloaddition reaction rather than nucleophilic substitution, so albumen can high selectivity be modified.By in reaction mixture, adding Cu (I) salt of catalytic amount, can at room temperature under aqueous conditions, carry out this reaction with the splendid locational choice (1,4＞1,5).For example referring to people such as Tornoe, (2002)Org.Chem.People such as 67:3057-3064 and Rostovtsev, (2002)Angew.Chem.Int.Ed.41:2596-2599.Spendable another kind of method is the dentate exchange that has on two arsenate compounds of four halfcystine primitives, for example referring to people such as Griffin, (1998)Science281:269-272.

Can comprise almost any molecule by the molecule that [3+2] cycloaddition reaction adds on the albumen of the present invention with nitrine or alkynyl derivatives.Molecule includes, but is not limited to dyestuff, fluorophore, linking agent, sugar derivatives, polymkeric substance (including, but is not limited to the derivative of polyoxyethylene glycol), photocrosslinking agent, cytotoxic compound, affinity labelling, biotin derivative, resin, bead, second albumen or polypeptide (or more), polynucleotide (including, but is not limited to DNA, RNA etc.), metal chelator, cofactor, lipid acid, carbohydrate or the like.These molecules can add to respectively on the alpha-non-natural amino acid of have alkynyl (including, but is not limited to alkynes propoxy-phenylalanine) or azido-(including, but is not limited to azido--phenylalanine).

V. produce the hGH polypeptide that comprises the non-genomic amino acids coding in the body

Can use modified tRNA and tRNA synthetic enzyme to produce hGH polypeptide of the present invention in vivo is not amino acids coding in the system of natural generation to add or to replace.

For example, use is not that the method that amino acids coding produces tRNA and tRNA synthetic enzyme in the system of natural generation is described in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931), it is incorporated herein by reference.These methods comprise producing and do not rely on the endogenous synthetic enzyme of translation system and tRNA (and thereby be sometimes referred to as " orthogonal ") and the body translation of functionating.Translation system comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) usually.Usually, O-RS produces with at least a non-natural preferably that amino acid aminoacylation O-tRNA and O-tRNA identification are at least a can't help the selection codon that other tRNA discerned in the system in translation system.Therefore translation system is reacted coded selection codon and with in the albumen that is produced in the non-naturally encoded amino acids insertion system, thus amino acid " replacement " is gone in the position in the encoded polypeptide.

Multiple quadrature tRNA and aminoacyl tRNA synthetase have been described in and are used for specific synthesizing amino acid is inserted in the technical field of polypeptide, and are applicable to the present invention usually.For example, ketone group specificity O-tRNA/ aminoacyl-tRNA synthetase is described in Wang, people such as L., and Proc.Natl.Acad.Sci.USA 100:56-61 (2003) and Zhang, people such as Z. are among Biochem.42 (22): the 6735-6746 (2003).Exemplary O-RS or its part are by polynucleotide sequence coding and comprise the aminoacid sequence that is disclosed in the U.S. Patent Application Publication case 2003/0082575 and 2003/0108885 (being incorporated herein by reference separately).Also be described in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) with the corresponding O-tRNA molecule of a use of O-RS, it is incorporated herein by reference.

The case description of nitrine specificity O-tRNA/ aminoacyl-tRNA synthetase system is in Chin, and people such as J. are among the J.Am.Chem.Soc.124:9026-9027 (2002).Be used for the exemplary O-RS sequence of azido--L-Phe is included, but is not limited to be disclosed in U.S. Patent Application Publication case 2003/0108885 (sequence number 10/126,931) nucleotide sequence SEQ ID NO:14-16 in and 29-32 and aminoacid sequence SEQ ID NO:46-48 and 61-64, described open case is incorporated herein by reference.Be applicable to that exemplary O-tRNA sequence of the present invention includes, but is not limited to be disclosed in U.S. Patent Application Publication case 2003/0108885 (sequence number 10/126, No. 931) in nucleotide sequence SEQ ID NO:1-3, described open case is incorporated herein by reference.Specific non-naturally encoded amino acids is other right case description of specific O-tRNA/ aminoacyl-tRNA synthetase in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927), and it is incorporated herein by reference.The amino acid whose O-RS and the O-tRNA that incorporate ketone group containing and azido-in the yeast saccharomyces cerevisiae (S.cerevisiae) are described in Chin, and people such as J. are among the Science 301:964-967 (2003).

The use of O-tRNA/ aminoacyl-tRNA synthetase comprises the specificity codon of selecting the coding non-naturally encoded amino acids.Though can use any codon, wish to be chosen in few using or no codon in the cell of expressing the O-tRNA/ aminoacyl-tRNA synthetase usually.For example, exemplary codon comprise nonsense codon such as terminator codon (amber, ochre and milky white), four or more polybase base codon and other few with or no natural three base codons.

Can use in affiliated field known sudden change to bring out method (include, but is not limited to that the locus specificity sudden change is brought out, cassette mutagenesis brings out, limit and select sudden change to bring out etc.) and specificity be selected the appropriate location in the codon introducing hGH polypeptid coding sequence.

Be used to produce the protein biology combination mechanism that can be used for incorporating into non-naturally encoded amino acids component (such as O-RSs, O-tRNAs and quadrature O-tRNA/O-RS to) method be described in Wang, people such as L., Science 292:498-500 (2001); Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002); Zhang, people such as Z. are among the Biochemistry 42:6735-6746 (2003).The method and composition that is used for incorporating in the body non-naturally encoded amino acids is described in the U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927), and it is incorporated herein by reference.The right method of quadrature tRNA-tRNA synthetic enzyme that is used to select to be used for translation system in the organic body also is described in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) in, it is incorporated herein by reference.

The method that is used to produce at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) comprises: (a) produce (being mutant according to circumstances) the RSs library that is derived from from the first organic at least a aminoacyl-tRNA synthetase (RS), described organism includes, but is not limited to such as Methanococcus jannaschii, thermophilic methagen, halophilic bacterium, intestinal bacteria (Escherichia coli), have a liking for ultrahigh-temperature sulfate radical reduction archeobacteria (A.fulgidus), fierce fireball bacterium (P.furiosus), Huo Shi fireball bacterium (P.horikoshii), hyperthermophilic archaeon strain (A.pernix), protokaryon organism of thermus thermophilus (T.thermophilus) or the like or eucaryon organism; (b) select in (and/or screening) RSs (be mutant RSs according to circumstances) library the member of aminoacylation quadrature tRNA (O-tRNA) in the presence of non-naturally encoded amino acids and natural amino acid, the set of activity (being mutant according to circumstances) RSs is provided thus; And/or (c) select in (according to circumstances by negative selection) described set there be not in the presence of the non-naturally encoded amino acids the preferably active RSs of aminoacylation O-tRNA (including, but is not limited to mutant RSs); at least a reorganization O-RS is provided thus, and wherein said at least a reorganization O-RS is preferably with non-naturally encoded amino acids aminoacylation O-tRNA.

In one embodiment, RS is nonactive RS.Nonactive RS can be produced by making active RS sudden change.For example, nonactive RS can by make at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or at least about 10 or more amino acids sport different aminoacids (including, but is not limited to L-Ala) and produced.

Under mutant RSs library can be used in the field known various technology produce, include, but is not limited to based on the appropriate design of protein three-dimensional RS structure or with at random or the appropriate design technology bring out the RS coding mutation.For example, the diversity that mutant RSs can be by locus specificity sudden change, random mutation, generation recombination mutation, chimeric construct that body, appropriate design and other this paper describe or affiliated field in known method produced.

In one embodiment, select in (and/or screening) RSs (being mutant RSs according to circumstances) library (including, but is not limited to) but in the presence of non-naturally encoded amino acids and natural amino acid the member of aminoacylation quadrature tRNA (O-RNA), it comprises: the positive selection or the selection markers that will include, but is not limited to antibiotics resistance gene or the like are introduced in a plurality of cells with (being mutant according to circumstances) RSs library, and wherein said positive selection and/or selection markers comprise the selection codon that at least one includes, but is not limited to amber, ochre or opal codon; Selecting the described a plurality of cells of growth in the presence of the medicament; By suppress positive select or selection markers at least one select codon to be identified in described selection and/or there is the cell of (or showing specific reaction) of survival down in the screening medicament, provide thus contain activity (being mutant according to circumstances) RS set select the subclass of cell through the positive.Select and/or screen drug concentration and can change according to circumstances.

On the one hand, positive selectable marker is that E.C. 2.3.1.28 (CAT) gene and selection codon are the amber terminator codon in the CAT gene.Positive selectable marker is that β-Nei Xiananmei gene and selection codon are the amber terminator codon in the described beta lactamase gene according to circumstances.On the other hand, the positive-selecting mark comprises fluorescence or luminous selection markers or based on the selection markers (including, but is not limited to cell surface marker) of avidity.

In one embodiment, negative select or screen in the described set do not having in the presence of the non-naturally encoded amino acids the preferably active RS of aminoacylation O-tRNA (being mutant according to circumstances), it comprises: feminine gender selection or selection markers are introduced in a plurality of second organism cells with the set of the activity of selecting from the positive or screen (being mutant according to circumstances) RSs, wherein said negative selection or selection markers comprise at least one and select codon (include, but is not limited to antibiotics resistance gene, include, but is not limited to E.C. 2.3.1.28 (CAT) gene); And be identified in survival in first substratum that is supplemented with non-naturally encoded amino acids and screening or selects medicament or show the specificity screening reaction but can not be in second substratum that is not supplemented with non-naturally encoded amino acids and selection or screening medicament survival or show the cell of specific reaction, survivaling cell or screening cell with at least one reorganization O-RS are provided thus.For example, the CAT identifying schemes serves as positive the selection and/or negative screening according to circumstances in the determining of suitable O-RS recombinant chou.For example, clone's set is duplicated on the growth flat board that contains the CAT (it comprises at least one and selects codon) that has or do not have one or more non-naturally encoded amino acids according to circumstances.Therefore think and contain reorganization O-RS containing on the flat board of non-naturally encoded amino acids alone the group of growth.On the one hand, select the concentration change of (and/or screening) medicament.In some respects, the described first and second organism differences.Therefore, described first and/or second organism comprises according to circumstances: prokaryotic organism, eukaryote, Mammals, intestinal bacteria, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc.In other embodiments, selection markers comprises fluorescence or luminous selection markers or based on the selection markers of avidity.

In another embodiment, activity (the being mutant according to circumstances) RSs in the described set of screening or selection (including, but is not limited to negative the selection) comprises: select step (b) to separate from the positive set of active mutant RSs; The set of feminine gender selection or selection markers and activity (being mutant according to circumstances) RSs is introduced in a plurality of second organism cells, wherein said negative selection or selection markers comprise at least one and select codon (to include, but is not limited to the toxicity marker gene, include, but is not limited to rnase barnase gene, it comprises at least one and selects codon); And be identified in first substratum that is not supplemented with non-naturally encoded amino acids survival or show the specificity screening reaction but in being supplemented with second substratum of non-naturally encoded amino acids, can not surviving or show the cell that specificity screening reacts, survival or screening cell with at least one reorganization O-RS are provided thus, and wherein said at least one reorganization O-RS has specificity to described non-naturally encoded amino acids.On the one hand, described at least one selection codon comprises two or more selection codons approximately.Described embodiment can comprise that according to circumstances wherein said at least one selection codon comprises two or more and selects codon, and the wherein said first and second organism differences (include, but is not limited to each organism and include, but is not limited to prokaryotic organism, eukaryote, Mammals, intestinal bacteria, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc. according to circumstances).Equally, some aspects comprise that wherein said negative selection marker comprises rnase barnase gene (it comprises at least one and selects codon).Others comprise that wherein said selection markers comprises fluorescence or luminous selection markers according to circumstances or based on the selection markers of avidity.In the embodiment of this paper, described screening and/or the variation of selecting to comprise screening according to circumstances and/or selecting preciseness.

In one embodiment, the method that is used to produce at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) can further comprise: (d) separate described at least a reorganization O-RS; (e) produce second group of O-RS (according to circumstances through sudden change) that is derived from described at least a reorganization O-RS; (f) repeating step (b) and (c) up to obtaining to comprise the preferably sudden change O-RS of the ability of the described O-tRNA of aminoacylation.Repeating step (d)-(f) includes, but is not limited at least about twice according to circumstances.On the one hand, can bring out (include, but is not limited to that random mutation brings out, locus specificity sudden change bring out), reorganization or its by sudden change makes up and produces described second group of sudden change O-RS that is derived from least a reorganization O-RS.

Include, but is not limited to positive selections/screening step (b), negative selections/screening step (c) or the positive and feminine gender selection/screening step (b) and (c) preciseness of both selection/screening steps in aforesaid method, comprise change selection/screening preciseness according to circumstances.In another embodiment, positive selections/screening step (b), negative selections/screening step (c) or positive and negative selection/screening step (b) and (c) both comprise use and report sub, wherein said report are to choose (FACS) by fluorescence activated cell to detect or wherein said report is to be detected by luminous.Displaying is reported son and is selected based on avidity that relates to non-naturally encoded amino acids or analogue or catalytic activity on cell surface, phage display or the like according to circumstances.In one embodiment, the synthetic enzyme of on cell surface, phage display or the like, showing sudden change.

The method that is used for generation reorganization quadrature tRNA (O-tRNA) comprises: (a) produce the mutant tRNAs library that is derived from from the first organic at least a tRNA (including, but is not limited to suppress tRNA); (b) select (including, but is not limited to negative the selection) or screen in the described library, tRNAs is provided the set of (being mutant according to circumstances) thus by (being mutant according to circumstances) tRNAs from the second organic aminoacyl-tRNA synthetase (RS) aminoacylation in the presence of not from the first organic RS; And (c) select or screening tRNAs (be mutant according to circumstances) set in by the member of quadrature RS (O-RS) aminoacylation of introducing; provide at least a reorganization O-tRNA thus, wherein said at least a reorganization O-tRNA identification selection codon and can't help from effectively identification and of the second organic RS preferably by described O-RS aminoacylation.In certain embodiments, described at least a tRNA is the uniqueness three base codons that suppress tRNA and/or comprise natural and/or non-natural base, perhaps is nonsense codon, rare codon, non-natural codon, the codon that comprises at least 4 bases, amber codon, ocher codon or opal temination codon.In one embodiment, the improvement of reorganization O-tRNA with orthogonality.Should be appreciated that, in certain embodiments, according to circumstances O-tRNA is imported first organism and need not modification from second organism.In various embodiments, first and second organisms are identical or different and be selected from (including, but is not limited to) prokaryotic organism (including, but is not limited to Methanococcus jannaschii, thermophilic methagen, intestinal bacteria, halophilic bacterium etc.), eukaryote, Mammals, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc. according to circumstances.In addition, tRNA is according to circumstances by the non-naturally encoded amino acids aminoacylation in reorganization, and wherein said non-naturally encoded amino acids is biosynthesizing or the biosynthesizing by genetic manipulation natively in vivo.According to circumstances non-naturally encoded amino acids is added in the growth medium to be used for first or second organism at least.

On the one hand, (being mutant according to circumstances) tRNAs (step (b)) by the aminoacyl-tRNA synthetase aminoacylation in selection (including, but is not limited to negative the selection) or the screening library comprising: introducing toxicity marker gene, wherein said toxicity marker gene comprises at least a of described selection codon and (or causes the gene of toxigenicity agent or electrostatic agent or be essential gene to described organism, wherein said marker gene comprises at least a selection codon), and introduce (being mutant according to circumstances) tRNAs library a plurality of from the second organic cell; And the selection survivaling cell, wherein said survivaling cell contains the set that comprises at least a quadrature tRNA or do not have (being mutant according to circumstances) tRNAs of function tRNA.For example, can select survivaling cell by using the compa-ratios cell density to examine and determine.

On the other hand, the toxicity marker gene can comprise two or more selection codons.In another embodiment of present method, the toxicity marker gene is a rnase barnase gene, and wherein said rnase barnase gene comprises at least one amber codon.Rnase barnase gene can comprise two or more amber codons according to circumstances.

In one embodiment, select or screening (being mutant according to circumstances) tRNAs set in can comprise by the member of quadrature RS (O-RS) aminoacylation of introducing: the positive is selected or selection markers gene and (being mutant according to circumstances) tRNAs set are introduced a plurality of from the second organic cell with O-RS, wherein said positive mark's gene comprises drug resistance gene and (includes, but is not limited to the β-Nei Xiananmei gene, it comprises at least one and selects codon, such as at least one amber terminator codon) or described organism is essential gene or makes toxic agents toxicide gene; And be identified in the survival that includes, but is not limited to growth under antibiotic selection or the existence of screening medicament or screen cell; cell aggregation with at least a reorganization tRNA is provided thus, and wherein said at least a reorganization tRNA is by described O-RS aminoacylation and replys described at least one selection codon and with in the translation product of aminoacid insertion by positive mark's genes encoding.In another embodiment, select and/or screen the concentration change of medicament.

Be provided for producing the right method of specificity O-tRNA/O-RS.Method comprises: (a) produce the library that is derived from from the mutant tRNAs of the first organic at least a tRNA; (b) negative select or the screening library in the presence of not from the first organic RS by (being mutant according to circumstances) tRNAs from second organic aminoacyl-tRNA synthetase (RS) aminoacylation, the set of (being mutant according to circumstances) tRNAs is provided thus; (c) select or screen in described (according to circumstances for mutant) tRNAs set, at least a reorganization O-tRNA is provided thus by the member of quadrature RS (O-RS) aminoacylation of introducing.Described at least a reorganization O-tRNA identification selection codon and can't help from effectively identification and of the second organic RS preferably by the O-RS aminoacylation.Described method comprises that also (d) produces (being mutant according to circumstances) the RSs library that is derived from from the 3rd organic at least a aminoacyl-tRNA synthetase (RS); (e) select or screen in the described mutant RSs library in the presence of non-naturally encoded amino acids and natural amino acid the preferably member of the described at least a reorganization O-tRNA of aminoacylation, the set of active (being mutant according to circumstances) RS is provided thus; (f) negatively select or screen in the described set do not having in the presence of the non-naturally encoded amino acids the preferably activity of the described at least a reorganization O-tRNA of aminoacylation (being mutant according to circumstances) RSs; provide described at least a specificity O-tRNA/O-RS right thus, wherein said at least a specificity O-tRNA/O-RS at least aly is specific reorganization O-RS and described at least a reorganization O-tRNA to non-naturally encoded amino acids to comprising.Comprise that the specificity O-tRNA/O-RS that is produced by described method is right.For example, described specificity O-tRNA/O-RS is right to comprising (including, but is not limited to) mutRNATyr-mutTyrRS, such as mutRNATyr-SS12TyrRS to, mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS to or the like.In addition, these methods comprise wherein said first identical with the 3rd organism (including, but is not limited to Methanococcus jannaschii).

Select quadrature tRNA-tRNA synthetic enzyme to also being included in the present invention with the method that is used for translation system in the second organic body.Described method comprises: marker gene, tRNA and the aminoacyl-tRNA synthetase (RS) that separates or be derived from it from first organism are introduced first group from the second organic cell; Described marker gene and tRNA are introduced from the second organic replicating cell group; And be chosen in the replicating cell group nonviable in first group survivaling cell or the cell of the screening displaying specificity screening reaction that in the replicating cell group, can not produce specificity screening reaction, wherein said first group and the growth in the presence of selection or screening medicament of replicating cell group, it is right that wherein said survival or screening cell comprise the quadrature tRNA-tRNA synthetic enzyme that is used for translation system in the second organic body.In one embodiment, relatively and select or screening comprises complementary calibrating in the body.But described selection and/or the change of screening drug concentration.

Organism of the present invention comprises multiple organism and multiple combination.For example, first and second organisms of method of the present invention can be identical or different.In one embodiment, described organism is the protokaryon organism according to circumstances, includes, but is not limited to Methanococcus jannaschii, thermophilic methagen, halophilic bacterium, intestinal bacteria, has a liking for ultrahigh-temperature sulfate radical reduction archeobacteria, fierce fireball bacterium, Huo Shi fireball bacterium, hyperthermophilic archaeon strain, thermus thermophilus or the like.Perhaps, described organism comprises the eucaryon organism according to circumstances, includes, but is not limited to plant (including, but is not limited to such as the complicated plant such as monocotyledons or dicotyledons), algae, protobiont, fungi (including, but is not limited to yeast etc.), animal (including, but is not limited to Mammals, insect, arthropods etc.) or the like.In another embodiment, described second organism is the protokaryon organism, includes, but is not limited to Methanococcus jannaschii, thermophilic methagen, halophilic bacterium, intestinal bacteria, has a liking for ultrahigh-temperature sulfate radical reduction archeobacteria, halophilic bacterium, fierce fireball bacterium, Huo Shi fireball bacterium, hyperthermophilic archaeon strain, thermus thermophilus or the like.Perhaps, described second organism can be the eucaryon organism, includes, but is not limited to yeast, zooblast, vegetable cell, fungi, mammalian cell or the like.In various embodiments, the described first and second organism differences.

VI. non-natural produces the position of amino acid in the hGH polypeptide

The present invention is contained and one or more non-naturals are produced amino acid is incorporated in the hGH polypeptide.One or more non-naturals produce amino acid can be incorporated at the specific position that does not destroy polypeptide active.It can replace (include, but is not limited to hydrophobic amino acid replacement hydrophobic amino acid, with the huge amino acid of huge aminoacid replacement, replace hydrophilic amino acid with hydrophilic amino acid) by carrying out " conservative property " and/or not realize for insertion non-natural in the active desired position produces amino acid.

Can be following the zone of explanation hGH, in the middle of wherein the amino acid position among the hGH is illustrated in the row (SEQ ID NO:2):

Spiral A spiral B spiral C spiral D

[1-5]-[6-33]-[34-74]-[75-96]-[97-105]-[106-129]-[130-153]-[154-183]-[184-191]

N-terminal A-B ring B-C ring C-D ring C-terminal.

Can use multiple biological chemistry and structural approach the site of being wanted in the hGH polypeptide, to select to be used for to replace with non-naturally encoded amino acids.One of ordinary skill in the art are easy to understand, and any position of polypeptide chain is suitable for selecting incorporating non-naturally encoded amino acids into, and for any or nonspecific institute syllabus, and selection can be carried out based on appropriate design or by selecting at random.The selection in the site of wanting can be used for producing having anyly to be wanted the hGH molecule of characteristic or activity (include, but is not limited to agonist, super agonist, reverse agonist, antagonist, receptors bind conditioning agent, receptor activity modulators, dimer or polymer formation, specific activity or characteristic do not have change mutually with natural molecule) or is used to handle any physics or chemical property such as solubleness, gathering or stable polypeptide.For example, under the position in the required polypeptide of the biological activity of hGH polypeptide can be used in the field known L-Ala scanning or homologue scan method discern.For example referring to Cunningham, B. and Wells, J., Science, 244:1081-1085 (1989) (identification is for 14 residues of hGH biological activity key) and Cunningham, B. wait the people, Science 243:1330-1336 (1989) (use homologue scanning sudden change to bring out and discern antibody and receptor antigen decision position).Except bring out by L-Ala or homologue scanning sudden change be identified as to the residue the residue of biological activity key for depend on for polypeptide seek want activity to be substituted by good candidate with non-naturally encoded amino acids.Perhaps, depend on once more by what polypeptide was sought and wanted active that the site that is identified as the biological activity key also is good candidate for replacing with non-naturally encoded amino acids.Another alternative method is for replacing simply continuously with non-naturally encoded amino acids in each position on polypeptide chain and observing effect to polypeptide active.Those of ordinary skill in the field are easy to understand, and any way, technology or the method that are used for selecting to be used for so that alpha-non-natural amino acid replaces into the position of any polypeptide are applicable to the present invention.

Also can detect the structure of mutant of natural generation of the hGH polypeptide that contains disappearance and active to determine the to allow albumen zone that replaces with non-naturally encoded amino acids.About hGH for example referring to people such as Kostyo, Biochem.Biophys.Acta, 925:314 (1987); Lewis, U. waits the people, J.Biol.Chem., 253:2679-2687 (1978).In a similar manner, can use the responsible hGH zone of protease digestion and monoclonal antibody identification in conjunction with the hGH polypeptide receptor.For example referring to Cunningham, people such as B., Science 243:1330-1336 (1989); Mills, people such as J., Endocrinology, 107:391-399 (1980); Li, C, Mol Cell.Biochem., 46:31-41 (1982) (showing that the amino acid between the residue 134-149 can lack and the non-activity loss).In case remove the residue that may not tolerate for the replacement of carrying out with non-naturally encoded amino acids, then can detect the proposed influence that is substituted on each rest position from hGH and protein-bonded three-dimensional crystalline structure thereof.About hGH please referring to de Vos, people such as A., Science, 255:306-312 (1992); All hGH crystalline structure all can obtain in the centralized data base albumen data bank (Protein Data Bank) (comprising 3HHR, 1AXI and 1HWG) (PDB can obtain on World Wide Web rcsb.org) of the macromole three-dimensional structure data that contains albumen and nucleic acid.Therefore, the those skilled in the art can be easy to discern the amino acid position that can non-naturally encoded amino acids replaces.

In certain embodiments, the hGH polypeptide of the present invention non-natural that comprises one or more albumen zones that are arranged in the spiral that do not destroy polypeptide or βZhe Die secondary structure produces amino acid.

The exemplary residue of incorporating non-naturally encoded amino acids into can be the residue that is excluded outside potential receptors bind zone (including, but is not limited to site I and site II), can be the residue that is exposed to solvent wholly or in part, have minimum or do not have interaction of hydrogen bond with near residue, but minimum exposure is near reactive residue, and can be on highly flexible (include, but is not limited to C-D ring) or structure in the zone of rigidity (including, but is not limited to the B spiral), as by as described in the three-dimensional crystalline structure of hGH polypeptide and its acceptor predict.

In certain embodiments, one or more non-naturally encoded amino acids be incorporated into corresponding to the secondary structure among the following hGH with one or more any position in the lower area: 1-5 (N-terminal), the 6-33 (A spiral) of SEQ ID NO:2, the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-terminal).In other embodiments, hGH polypeptide of the present invention comprises and replaces amino acid whose at least one non-natural generation amino acid that at least one is arranged at least one hGH zone, and described zone is to be selected from the group that is made up of N-terminal, B-C ring (97-105), C-D ring (132-149) and the C-terminal (184-191) of N-terminal (1-5), A-B ring (32-46).In certain embodiments, one or more non-naturally encoded amino acids be incorporated into hGH with in one or more of upper/lower positions: before the position 1 (being N-terminal), 1,2,3,4,5,6,7,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).

The exemplary site of incorporating one or more non-naturally encoded amino acids into comprises corresponding amino acid whose 29 of SEQ ID NO:2 or SEQ IDNO:1 or 3,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 or its any combination.

The subclass that is used to incorporate into the exemplary site of one or more non-naturally encoded amino acids comprises corresponding amino acid whose 29 of SEQ ID NO:2 or SEQ ID NO:1 or 3,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 or its any combination.The side chain that shows these amino-acid residues to the crystalline structure of hGH and with the interactional detection of hGH acceptor wholly or in part can by solvent near and the side chain of non-naturally encoded amino acids can stretch out and enter the solvent from protein surface.

The exemplary position that is used to incorporate into one or more non-naturally encoded amino acids comprises corresponding amino acid whose 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Show that to the crystalline structure of hGH and with the interactional detection of hGH acceptor the side chain that the side chain of these amino-acid residues is exposed to solvent and primary residue fully stretches out to enter solvent.

The exemplary site subclass that is used to incorporate into one or more non-naturally encoded amino acids comprises corresponding amino acid whose 30,74,103 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.Another subclass that is used to incorporate into the exemplary site of one or more non-naturally encoded amino acids comprises corresponding amino acid whose 35,92,143,145 or its any combination of SEQ ID NO:2 or SEQ ID NO:1 or 3.

In certain embodiments, non-natural in one or more of these positions produces amino acid and is connected in water-soluble polymers, includes, but is not limited to upper/lower positions: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (the corresponding amino acid of SEQ ID NO:2 or SEQID NO:1 or 3).In certain embodiments, the non-natural in one or more of these positions produces amino acid and is connected in water-soluble polymers: 30,35,74,92,103,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQ ID NO:1 or 3).In certain embodiments, the non-natural in one or more of these positions produces amino acid and is connected in water-soluble polymers: 35,92,143,145 (the corresponding amino acid of SEQ ID NO:2 or SEQID NO:1 or 3).

Human GH antagonist include, but is not limited to those in 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 and 127 have replace or in position 1 (being N-terminal) have add or its any combination (SEQ ID NO:2 or SEQ ID NO:1,3 or any other GH sequence in corresponding amino acid) antagonist.

Multiple non-naturally encoded amino acids can be replaced or incorporates into the given position in the hGH polypeptide.Generally speaking; detection based on the three-dimensional crystalline structure of hGH polypeptide and its acceptor; to conservative property replace preferred (promptly such as non-naturally encoded aminoacid replacement Phe based on aryl to acetyl phenyl alanine or O-propargyl tyrosine; Tyr or Trp) and people's specific conjugation of wishing to introduce in the hGH polypeptide (for example engage chemistry; if people wish to realize Huisgen[3+2 with the water-soluble polymers with alkynyl moiety] cycloaddition; then introduce 4-triazobenzene L-Ala; if perhaps people wish to form amido linkage with the water-soluble polymers with aryl ester, then then incorporate the phosphine part into) select specific non-naturally encoded amino acid to incorporate into being used to.

In one embodiment, described method further comprises to be incorporated alpha-non-natural amino acid in the albumen into, and wherein said alpha-non-natural amino acid comprises first reactive group; And (include, but is not limited to mark with the molecule that comprises second reactive group, dyestuff, polymkeric substance, water-soluble polymers, polyethyleneglycol derivative, the photocrosslinking agent, cytotoxic compound, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, NDA, RNA, antisense polynucleotides, inhibition Yeast Nucleic Acid, biomaterial, nano particle, spin labeling, fluorophore, the containing metal part, radioactive segment, novel functional group, with the covalently or non-covalently interactional group of other molecule, the light enclosure portion, but the part of photoisomerization, vitamin H, biotin derivative, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, prolong side chain, the sugar that is connected with carbon, redox active agent, amino thioic acid sulfoacid, the toxicity part, isotope-labeled part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable label, any combination of small molecules or above material, perhaps any other wanted compound or material) contact described albumen.First reactive group and the reaction of second reactive group are to be connected in alpha-non-natural amino acid with molecule by [3+2] cycloaddition reaction.In one embodiment, first reactive group is that alkynyl or the azido-part and second reactive group are azido-or alkynyl part.For example, first reactive group is that the alkynyl part (include, but is not limited to alpha-non-natural amino acid to alkynes propoxy-phenylalanine in) and second reactive group are the azido-part.In another example, first reactive group is that the azido-part (include, but is not limited to alpha-non-natural amino acid to azido--L-phenylalanine in) and second reactive group are the alkynyl part.

In some cases, non-naturally encoded amino acids replace with the hGH polypeptide in other add, replace or the disappearance combination to influence other biological characteristics of hGH polypeptide.In some cases, described other adds, replaces or lack the stability (including, but is not limited to the resistance to the degraded of proteolysis type) that can increase the hGH polypeptide or increase the avidity of hGH polypeptide to its acceptor.In certain embodiments, the hGH polypeptide comprises F10A, F10H, the F10I that is selected from by among the SEQ ID NO:2; M14W, M14Q, M14G; H18D; H21N; G120A; R167N; D171S; E174S; The aminoacid replacement of the group that F176Y, I179T or its any combination are formed.In some cases, described other adds, replaces or disappearance can increase the solubleness (including, but is not limited to when expressing) of hGH polypeptide in intestinal bacteria or other host cell.In certain embodiments, in the intestinal bacteria recombinant host cell, express interpolation afterwards, replacement or disappearance and can increase the polypeptide solvability.In certain embodiments, except that being used to incorporate into another site of alpha-non-natural amino acid, select the site to replace with natural amino acids coding or alpha-non-natural amino acid, it increases the polypeptide solvability after making and expressing in the intestinal bacteria recombinant host cell.In certain embodiments, the hGH polypeptide comprises another interpolation, replacement or disappearance, and it is regulated avidity, adjusting (including, but is not limited to increases or reduce) receptor dimerization effect to the hGH polypeptide receptor, stablize receptor dimer, adjusting circulating half-life, adjustment release or biological utilisation, helps purifying or improvement or change specific dosing way.For example, except one or more non-naturally encoded amino acids of introducing as described herein, introduce one or more following replacements: F10A, F10H or F10I; M14W, M14Q or M14G; H18D; H21N; R167N; D171S; E174S; F176Y and I179T are to increase the avidity of hGH varient to its acceptor.Similarly, the hGH polypeptide can comprise protease cracking sequence, reactive group, antibody binding domain (including, but is not limited to FLAG or poly-His) or other sequence based on avidity (including, but is not limited to FLAG, poly-His, GST etc.) or the molecule (including, but is not limited to vitamin H) through connecting that the improvement polypeptide detects (including, but is not limited to GFP), purifying or further feature.

In certain embodiments, the replacement of non-naturally encoded amino acids produces the hGH antagonist.The subclass that is used for incorporating into the exemplary site of one or more non-naturally encoded amino acids comprises: 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123,127 or the interpolation before 1 in the position (SEQ ID NO:2 or SEQ ID NO:1,3 or the corresponding amino acid of any other GH sequence).In certain embodiments, the hGH antagonist comprises at least one replacement that makes GH serve as antagonist in lower area: 1-5 (N-terminal), 6-33 (A spiral), the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-terminal).In other embodiments, the exemplary site of incorporating non-naturally encoded amino acids into is included in the amino terminal region of spiral A and the residue in the part spiral C.In another embodiment, use such as non-naturally encoded amino acids replacement G120 azido--L-phenylalanine or O-propargyl-L-tyrosine.In other embodiments, replacement listed above and the extra replacement that makes the hGH polypeptide become the hGH antagonist are made up.For example, on one of position of this paper identification, replace non-naturally encoded amino acids and introduce at G120 and to replace (for example G120R, G120K, G120W, G120Y, G120F or G120E) simultaneously.In certain embodiments, the hGH antagonist comprises the non-naturally encoded amino acids that is connected with water-soluble polymers in the receptor binding domain that is present in the hGH molecule.

In some cases, replace 1,2,3,4,5,6,7,8,9,10 or amino acids more with one or more non-naturally encoded amino acids.In some cases, the hGH polypeptide further comprises one or more non-naturally encoded amino acids to natural generation amino acid whose 1,2,3,4,5,6,7,8,9,10 or more polysubstituted.For example, in certain embodiments, at least two residues in following hGH zone replace through one or more non-naturally encoded amino acids: 1-5 (N-terminal); 32-46 (N-terminal of A-B ring); 97-105 (B-C ring); 132-149 (C-D ring); And 184-191 (C-terminal).In certain embodiments, at least two residues in following hGH zone replace through one or more non-naturally encoded amino acids: 1-5 (N-terminal), 6-33 (A spiral), the 34-74 (zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), the 97-105 (zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (zone between C spiral and the D spiral, C-D ring), 154-183 (D spiral), 184-191 (C-terminal).In certain embodiments, at least two residues in following hIFN zone replace through one or more non-naturally encoded amino acids: 1-9 (N-terminal), 10-21 (A spiral), 22-39 (zone between A spiral and the B spiral), 40-75 (B spiral), 76-77 (zone between B spiral and the C spiral), 78-100 (C spiral), 101-110 (zone between C spiral and the D spiral), 111-132 (D spiral), 133-136 (zone between D spiral and the E spiral), 137-155 (E spiral), 156-165 (C-terminal).In some cases, the residue that two or more are non-naturally encoded is connected in the straight chain of one or more lower molecular weights or branch PEG (overall pact～5-20kDa or littler), strengthens binding affinity and suitable serum half-life with respect to the material of the PEG that is connected in single higher molecular weight thus.

In certain embodiments, replacing up to two following hGH residue with one or more non-naturally encoded amino acids with upper/lower positions: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187.In some cases, form following right any one of replacing: K38X^*And K140X^*, K41X^*And K145X^*, Y35X^*And E88X^*, Y35X^*And F92X^*, Y35X^*And Y143X^*, F92X^*And Y143X^*, X wherein^*The expression non-naturally encoded amino acids.The preferred sites that is used to incorporate into two or more non-naturally encoded amino acids comprises the combination of following residue: 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187.The especially preferred sites that is used to incorporate into two or more non-naturally encoded amino acids comprises the combination of following residue: 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155.

The preferred sites that is used to incorporate into two or more non-naturally encoded amino acids in hGH comprises the combination of following residue: before theposition 1 of SEQ ID NO:2 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) or its any combinations.

VII. the expression in non-eukaryote and eukaryote

Be the high level expression of the hGH polynucleotide that obtain to be cloned, people will encode usually the polynucleotide subclone of hGH polypeptide of the present invention be used to instruct the strong promoter of transcribing to containing, transcribe/translation termination and (if for nucleic acid of proteins encoded) be used for the expression vector of the ribosome bind site of translation initiation.The bacterium promotor that is fit to is well-known in affiliated field and for example is described among the people such as people such as Sambrook and Ausubel.

The bacterial expression system that is used for expressing hGH polypeptide of the present invention can obtain (people such as Palva, Gene 22:229-235 (1983) at (including, but is not limited to) intestinal bacteria, bacillus (Bacillus sp.) and salmonella (Salmonella); People such as Mosbach, Nature 302:543-545 (1983)).The test kit that is used for these expression systems can be buied by the market.The eukaryotic expression system that is used for mammalian cell, yeast and insect cell is well-known in affiliated field and also can be buied by the market.Use quadrature tRNA and aminoacyl tRNA synthetase (the above) to express under the situation of hGH polypeptide of the present invention therein, use the ability of quadrature component to select the host cell that is used to express based on it.Exemplary host cell comprises that gram positive bacterium (Gram-positive bacteria) (includes, but is not limited to bacillus pumilus (B.brevis), subtilis (B.subtilis) or streptomyces (Streptomyces)) and gram negative bacterium (Gram-negative bacteria) (intestinal bacteria, Pseudomonas fluorescens (Pseudomonasfluorescens), Pseudomonas aeruginosa (Pseudomonas aerugiNOa), pseudomonas putida (Pseudomonas putida)) and yeast and other eukaryotic cell.Can use as described herein comprise the right cell of O-tRNA/O-RS.

Eukaryotic host cell of the present invention or non-eukaryotic host cell provide the synthetic proteic ability that comprises the alpha-non-natural amino acid of huge suitable quantity.On the one hand, composition comprises (including, but is not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or the albumen that comprises alpha-non-natural amino acid according to circumstances, perhaps can body in the amount (recombinant protein produces and the details of purifying is provided in herein) that realizes of process for generation of protein.On the other hand, described albumen is present in the composition with following concentration according to circumstances, and described concentration includes, but is not limited to including, but is not limited to cell lysates, damping fluid, whenever rising in medicine damping fluid or other liquid suspension (including, but is not limited to include, but is not limited to about 1nl to the volume between about 100L) lacked 10 microgram albumen, whenever rise to few 50 microgram albumen, whenever rise to few 75 microgram albumen, whenever rise to few 100 microgram albumen, whenever rise to few 200 microgram albumen, whenever rise to few 250 microgram albumen, whenever rise to few 500 microgram albumen, whenever rise to few 1 milligram of albumen or whenever rise to few 10 milligrams of albumen or more.In eukaryotic cell, produce a large amount of (including, but is not limited to can obtainable amount greater than using other to include, but is not limited in vitro translated method usually) and comprise that the albumen of at least a alpha-non-natural amino acid is a feature of the present invention.

Eukaryotic host cell of the present invention or non-eukaryotic host cell provide the proteic ability of the alpha-non-natural amino acid that biosynthesizing comprises huge suitable quantity.For example, can following concentration produce the albumen that comprises alpha-non-natural amino acid, described concentration includes, but is not limited at the cell extraction thing, cell lysates, substratum, at least 10 micrograms per litre in damping fluid or the like, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre, at least 1 mg/litre, at least 2 mg/litre, at least 3 mg/litre, at least 4 mg/litre, at least 5 mg/litre, at least 6 mg/litre, at least 7 mg/litre, at least 8 mg/litre, at least 9 mg/litre, at least 10 mg/litre, at least 20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900 mg/litre, 1 grams per liter, 5 grams per liters, 10 grams per liters or greater protein.

I. expression system, cultivation and separate

The hGH polypeptide can be expressed in any amount of suitable expression system, and described system comprises for example yeast, insect cell, mammalian cell and bacterium.The description of exemplary expression system is provided in hereinafter.

Yeast is as used herein, and term " yeast " comprises any in each primary yeast of the gene that can express coding hGH polypeptide.These yeast include, but is not limited to ascosporogenous yeast (Endomycetale (Endomycetales)), sporidium yeast and belong to the yeast of imperfect fungi (gemma guiding principle (Blastomycetes)) class.Described ascosporogenous yeast is divided into two sections, i.e. Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises four subfamilies, Schizosaccharomycoideae (for example Schizosaccharomyces (Schizosaccharomyces)), Nadsonioideae, Lipomycoideae and Saccharomycoideae (for example Pichia (Pichia), genus kluyveromyces (Kluyveromyces) and yeast belong (Saccharomyces)).The sporidium yeast comprises that Leucosporidium genus, Rhodosporidium (Rhodosporidium), lock are thrown yeast belong (Sporidiobolus), Filobasidium belongs to and Filobasidiella (Filobasidiella).The yeast that belongs to imperfect fungi (gemma guiding principle) class is divided into two sections, i.e. Sporobolomycetaceae (Sporobolomycetaceae) (for example Sporobolomyces (Sporobolomyces) and cloth are reined in and played spore yeast belong (Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example mycocandida (Candida)).

Being used for especially relevant species of the present invention is with the species in the subordinate: Pichia, genus kluyveromyces, yeast belong, Schizosaccharomyces, Hansenula (Hansenula), torulopsis (Torulopsis) and mycocandida, it includes, but is not limited to methanol yeast (P.pastoris), P.guillerimondii, yeast saccharomyces cerevisiae (S.cerevisiae), Ka Ersibai yeast (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), S.douglasii, S.kluyveri, S.norbensis, S.oviformis, Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), Candida albicans (C.albicans), maltose candiyeast (C.maltosa) and polymorphic debaryomyces hansenii (H.polymorpha).

The suitable yeast that selection is used to express the hGH polypeptide is in those of ordinary skill in the field's limit of power.At the yeast host of selecting to be used for expressing, appropriate host can comprise that displaying has for example good secretion capacity, low proteolytic activity, good secretion capacity, good soluble protein produces and overall firm yeast.Yeast can obtain from multiple source usually, it includes, but is not limited to inferior university's (Berkeley branch school) biophysics of Gary welfare and medicophysics is yeast heredity reserve center (Yeast Genetic Stock Center, Department of Biophysics and MedicalPhysics, University ofCalifornia (Berkeley, CA)) and U.S. typical case culture collecting center (AmericanType Culture Collection) (" ATCC ") (Manassas, VA).

Term " yeast host " or " yeast host cell " comprise the yeast that can be used as or be used as the receptor of recombinant vectors or other transfer DNA.Described term comprises the offspring of the original yeast host cell that receives recombinant vectors or other transfer DNA.Should be appreciated that because chance or the sudden change had a mind to, the offspring of single parental cell can on the form or with original parent complementary genome or total DNA in full accord.Being characterized as offspring such as the parental cell that fully is similar to the parent of the correlation properties of the nucleotide sequence that has coding hGH polypeptide is included among the offspring that this definition means.

The expression that comprises extrachromosomal replication or integrative vector and conversion carrier have been developed to be used for being transformed into multiple yeast host.For example, developed and be used for following zymic expression vector: yeast saccharomyces cerevisiae (people such as Sikorski, GENETICS (1998) 112:19; People such as Ito, J.BACTERIOL. (1983) 153:163; People such as Hinnen, PROC.N_ATL.A_CAD.S_CI.USA Candida albicans (people such as Kurtz (1978) 75:1929),, MOL.CELL.BIOL. maltose candiyeast (people such as Kunze (1986) 6:142),, J.BASIC MlCROBiOL. (1985) 25:141), polymorphic debaryomyces hansenii (people such as Gleeson, J.GEN.M_ICROBIOL. (1986) 132:3459; People such as Roggenkamp, M_OL.G_EN.G_ENET. (1986) 202:302), Kluyveromyces fragilis (people such as Das, J.BACTERIOL. (1984) 158:1165), Kluyveromyces lactis (people such as De Louvencourt, J.B_ACTERIOL. (1983) 154:737; People such as Van denBerg, B_IO/ T_ECHNOLOGY(1990) 8:135), P.guillerimondii (people such as Kunze, J.BASICMICROBIOL. (1985) 25:141), methanol yeast (United States Patent (USP) the 5th, 324,639,4,929,555 and 4,837, No. 148; People such as Cregg, MOL.CELL.BIOL. (1985) 5:3376), schizosaccharomyces pombe (Schizosaccharomycespombe) (Beach and Nurse, NATURE (1981) 300:706) conciliates the inferior sieve yeast (Y.lipolytica) of fat (people such as Davidow, C_URR.GENET. (1985) 10:380 (1985); People such as Gaillardin, CURR.GENET. (1985) 10:49), Aspergillus nidulans (A.nidulans) (people such as Balance, BlOCHEM.BlOPHYS.R_ES.COMMUN. (1983) 112:284-89; People such as Tilburn, G_ENE(1983) people such as 26:205-221 and Yelton, P_ROC.N_ATL.A_CAD.S_CI.USA (1984) 81:1470-74), aspergillus niger (A.niger) (Kelly and Hynes, EMBO J. (1985) 4:475479), Rui Shi wood mould (T.reesia) (EP 0 244 234) and filamentous fungus, such as neurospora (Neurospora), Penicillium notatum (Penicillium), curved neck mould (Tolypocladium) (WO 91/00357), it is incorporated herein by reference separately.

The control sequence that is used for yeast vector is well-known for the those of ordinary skill in affiliated field, and includes, but is not limited to from the promoter region such as the gene of following gene: alcoholdehydrogenase (ADH) (EP 0 284 044), Hydratase, phosphoenolpyruvate, glucokinase, G-6-P isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3-phoshoglyceric acid mutase and pyruvate kinase (PyK) (EP 0 329 203).The yeast PHO5 gene of coding acid phosphatase also can provide promoter sequence (people such as Myanohara, the P of usefulness_ROC.N_ATL.A_CAD.SCI.USA (1983) 80:1).Other suitable promoter sequence that is used for yeast host comprises and is used for glycerol 3-phosphate acid kinase (people such as Hitzeman, J.BIOL.C_HEM. (1980) 255:2073) and other glycolytic enzyme such as pyruvic carboxylase, triosephosphate isomerase and phosphoglucose isomerase (people such as Holland, B_IOCHEMISTRY(1978) 17:4900; People such as Hess, J.A_DV.E_NZYMER_EG. (1968) 7:149) promotor.Have induced Yeast promoter by the additional advantage of transcribing of growth conditions control and can comprise the promoter region of the enzyme that is used for alcoholdehydrogenase 2, different cell pigment C, acid phosphatase, metallothionein(MT), glyceraldehyde-3-phosphate dehydrogenase, the degrading enzyme relevant and responsible maltose and galactose utilization with nitrogen metabolism.The suitable carrier and the promotor that are used for yeast expression are further described in EP 0 073657.

The yeast enhanser also can use with Yeast promoter.In addition, synthetic promoter also can play the effect of Yeast promoter.For example, the upstream activating sequence of Yeast promoter (UAS) can be connected in the transcriptional activation domain of another Yeast promoter, thereby produces synthetic hybrid promoter.The example of these hybrid promoters comprises the ADH regulating and controlling sequence that is connected in the GAP transcriptional activation domain.Referring to United States Patent (USP) the 4th, 880,734 and 4,876, No. 197, it is incorporated herein by reference.Other example of hybrid promoter comprises the regulating and controlling sequence and the promotor of forming such as the glycolytic enzyme gene transcription active region of GAP or PyK by ADH2, GAL4, GAL10 or PHO5 gene.Referring toEP 0 164 556.In addition, Yeast promoter can comprise the promotor in the non-yeast source of the natural generation with combining yeast RNA polymerase and initial ability of transcribing.

Other controlling elements that can comprise the part Yeast expression carrier for example comprises terminator (people such as Holland, the J.BIOL.C from GAPDH or enolase gene_HEM. (1981) 256:1385).In addition, the replication orgin from 2 μ plasmid starting points is applicable to yeast.The suitable selection gene that is used for yeast is the trpl gene that is present in the yeast plasmid.Referring to people such as Tschemper, GENE (1980) 10:157; People such as Kingsman, GENE (1979) 7:141.Described trpl gene is provided for lacking the selective marker of the yeast mutant of the ability of growing in tryptophane.Similarly, replenish Leu2 defective yeast bacterial strain (ATCC 20,622 or 38,626) by known plasmid with Leu2 gene.

The method that foreign DNA is introduced in the yeast host is well-known to those of ordinary skill in the field, and generally includes (but being not limited to) conversion spheroplast or transform the complete yeast host cell of handling with alkaline kation.For example, yeast conversion can be carried out according to the method for describing in the following document: people such as Hsiao, P_ROC.N_ATL.A_CAD.S_CI.USA people such as (1979) 76:3829 and Van Solingen, J.B_ACT. (1977) 130:946.Yet, also can be as usually people such as SAMBROOK, M_OLECULARC_LONING: A L_AB.M_ANUAL(2001) described in, use such as other that merges by nuclear injection, electroporation or protoplastis to be used for DNA is introduced the method for cell.Can use the known standard technique culturing yeast of those of ordinary skill in the field host cell then.

Being used in proteic other method of yeast host cell expressing heterologous is well-known to those of ordinary skill in the field.Usually referring to No. the 20020055169th, U.S. Patent Publication case, United States Patent (USP) the 6th, 361,969,6,312,923,6,183,985,6,083,723,6,017,731,5,674,706,5,629,203,5,602,034 and 5,089, No. 398, U.S. check patent RE37,343 and RE35, No. 749, PCT publication application case WO 99/078621, WO 98/37208 and WO 98/26080, Europeanpatent application EP 0 946 736,EP 0 732 403,EP 0 480480,EP 0 460 071,EP 0 340 986,EP 0 329 203,EP 0 324 274 andEP 0 164 556.Also referring to people such as Gellissen, A_NTONIEV_ANL_EEUWENHOEK(1992) 62 (1-2): 79-93; People such as Romanos, Y_EAST(1992) 8 (6): 423-488; Goeddel, M_{ETHODS IN}E_NZYMOLOGY(1990) 185:3-7, it is incorporated herein by reference separately.

Can use those of ordinary skill in the field's well-known standard fed-batch fermentation methods (standardfeed batch fermentation method) that the yeast host bacterial strain is grown in fermentor tank in the amplification stage.Described fermentation method can utilize the path or express difference in the master mode through adjusting with the carbon that solves specific yeast host.For example, the fermentation of yeast belong yeast host may need single glucose feedback material, compound nitrogen source (for example caseic hydrolysate) and multivitamin to replenish.On the contrary, thermophilic methanol yeast fungus beetle alcohol yeast may need glycerine, methyl alcohol and trace mineral feedback material, but only needs simple ammonium (nitrogen) salt to be used for optimum growh and expression.For example referring to United States Patent (USP) the 5th, 324, people such as No. 639, Elliott, J.PROTEIN C_HEM. people such as (1990) 9:95 and Fieschko, BIOTECH.BIOENG. (1987) 29:1113, it is incorporated herein by reference.

Yet these fermentation process can have some the common feature that is independent of the used yeast host strain.For example, the growth limitation nutrient substance that is generally carbon can add in the fermentor tank to allow maximum growth in the amplification stage.In addition, the common Application Design of fermentation method is the fermention medium of the carbon that contains q.s, nitrogen, basic salt, phosphorus and other less important nutrient substance (for example VITAMIN, trace mineral and salt etc.).The case description of fermention medium that is applicable to pichia spp is in United States Patent (USP) the 5th, 324, and 639 and 5,231, in No. 178, it is incorporated herein by reference.

The insect cell term " insect host " of baculovirus infection or " insect host cell " mean the insect that can be used as or be used as the receptor of recombinant vectors or other transfer DNA.Described term comprises the offspring of the protentomon host cell of transfection.Should be appreciated that because chance or the sudden change had a mind to, the offspring of single parental cell can on the form or with original parent complementary genome or total DNA in full accord.Being characterized as offspring such as the parental cell that fully is similar to the parent of the correlation properties of the nucleotide sequence that has coding hGH polypeptide is included among the offspring that this definition means.

The suitable insect cell that selection is used to express the hGH polypeptide is well-known to those of ordinary skill in the field.Several insect species are fully described in affiliated field and can be buied by the market, and it comprises Aedes aegypti (Aedes aegypti), silkworm (Bombyx mori), drosophila melanogaster (Drosophila melanogaster), fall army worm (Spodopterafrugiperda) and cabbage looper (Trichoplusia ni).At the insect host of selecting to be used for expressing, appropriate host can comprise that displaying especially has the host of good secretion capacity, low proteolytic activity and general health.Insect can obtain from multiple source usually, it includes, but is not limited to inferior university's (Berkeley branch school) biophysics of Gary welfare and medicophysics is insect heredity reserve center (Insect Genetic Stock Center, Department of Biophysics andMedical Physics, University of California (Berkeley, CA)) and U.S. typical case culture collecting center (" ATCC ") (Manassas, VA).

The component of the insect expression system of baculovirus infection generally includes: be generally the transfer vector of bacterial plasmid, the convenient restriction site that it contains the baculovirus genomic fragment and is used to insert the heterologous gene that remains to be expressed; Wild-type baculovirus, its have with transfer vector in baculovirus specific fragment homologous sequence (its permission is gone into the heterologous gene homologous recombination in the baculovirus genome); With suitable insect host cell and growth medium.Be used for constructing carrier, transfectional cell, select bacterial plaque, material, method and the technology of the cell of grown culture or the like be known in affiliated field and can obtain to describe the handbook of these technology.

After heterologous gene is inserted transfer vector, carrier and wild-type virus genome are transfected in the insect host cell wherein said carrier and viral genome reorganization.Expression is through recombinant virus and the identification and the purification of Recombinant bacterial plaque of packing.Be used for the material of baculovirus/insect cell expression system and method can kit form (Carlsbad CA) buys from Invitrogen Corp. for example.It is known and be described in SUMMERS fully that these technology are generally the those skilled in the art_ANDS_MITH, T_EXASA_GRICULTURALE_XPERIMENTS_TATIONB_ULLETINNo.1555 (1987), it is incorporated herein by reference.Also referring to RICHARDSON, 39 METHODS INMOLECULAR BIOLOGY:B_ACULOVIRUSE_XPRESSIONP_ROTOCOLS(1995); A_USUBELDeng the people, C_URRENTP_{ROTOCOLS IN}M_OLECULARB_IOLOGY16.9-16.11 (1994); K_INGAnd P_OSSEE, T_HEB_ACULOVIRUSS_YSTEM: A L_ABORATORYG_UIDE(1992); And O ' R_EILLYDeng the people, B_ACULOVIRUSE_XPRESSIONV_ECTORS: A L_ABORATORYM_ANUAL(1992).

In fact, using baculovirus/insect cell expression system to produce various heterologous proteins is well-known in affiliated field.For example referring to United States Patent (USP) the 6th, 368,825,6,342,216,6,338,846,6,261,805,6,245,528,6,225,060,6,183,987,6,168,932,6,126,944,6,096,304,6,013,433,5,965,393,5,939,285,5,891,676,5,871,986,5,861,279,5,858,368,5,843,733,5,762,939,5,753,220,5,605,827,5,583,023,5,571,709,5,516,657,5,290, No. 686; WO 02/06305, WO 01/90390, WO 01/27301, WO 01/05956, WO 00/55345, WO 00/20032, WO 99/51721, WO 99/45130, WO 99/31257, WO 99/10515, WO 99/09193, WO 97/26332, WO 96/29400, WO 96/25496, WO 96/06161, WO 95/20672, WO 93/03173, WO 92/16619, WO 92/03628, WO 92/01801, WO 90/14428, WO 90/10078, WO 90/02566, WO 90/02186, WO 90/01556, WO 89/01038, WO 89/01037, WO 88/07082, and it is incorporated herein by reference.

Be applicable to that the carrier in baculovirus/insect cell expression system is known in affiliated field, and comprise that for example being derived from baculovirus autographa california nuclear polyhedrosis virus (baculovirus Autographacalifornica nuclearpolyhedrosis virus) insect (AcNPV) expresses and transfer vector, AcNPV is not for relying on the virus expression carrier of subsidiary.The virus expression carrier that is derived from this system uses the strong virus polyhedrin gene promoter to drive expression of heterologous genes usually.Usually referring to people such as Reilly, BACULOVIRUS EXPRESSION VECTORS:ALABORATORY MANUAL (1992).

Before alien gene was inserted shaft-like viral genome, the above-mentioned component that will comprise promotor, leader sequence (if desired), correlative coding sequence and transcription termination sequence usually was assembled into middle the exchange and constructs in the body (transfer vector).Middle exchange is constructed body and is remained in usually in the replicon, remains in such as the extra-chromosomal element among the host of bacterium (for example plasmid) such as stablizing.Replicon will have dubbing system, therefore it be remained in the appropriate host to clone and to increase.More particularly, plasmid can contain polyhedrin polyadenylation signal people such as (, ANN.REV.MiCROBiOL. (1988) 42:177) Miller and procaryotic amicillin resistance (amp) gene and replication orgin to be selected and breeding intestinal bacteria being used for.

The transfer vector a kind of commonly used that is used for alien gene is introduced AcNPV is pAc373.Known to the skilled multiple other carrier in field under also having designed, it for example comprises the polyhedrin initiator codon from ATG changes into ATT and 32 base pairs are introduced the BamHI cloning sites in the ATT upstream pVL985.Referring to Luckow and Summers, 17 VIROLOGY 31 (1989).Other commercially available carrier for example comprise PBlueBac4.5/V5-His, pBlueBacHis2, pMelBac, pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).

After inserting heterologous gene, transfer vector and wild-type baculovirus genome cotransfection are gone among the insect cell host.The method that is used for allogeneic dna sequence DNA is introduced the site of wanting of baculovirus is known in affiliated field.Referring to S_UMMERSAnd S_MITH, T_EXASA_GRICULTURALE_XPERIMENTS_TATIONB_ULLETINNo.1555 (1987); People such as Smith, M_OL.C_ELL.B_IOL. (1983) 3:2156; Luckow and Summers, V_IROLOGY(1989) 17:31.For example, described insertion can enter gene such as polyhedron gene by homology double exchange reorganization; Insert and also can enter through the restriction enzyme sites of engineering design for the baculovirus gene of wanting.Referring to people such as Miller, BlOESSAYS (1989) 4:91.

Can finish transfection by electroporation.Referring to TROTTER and WOOD, 39 M_{ETHODS IN}M_OLECULARB_IOLOGY(1995); Mann and King, J.G_EN.V_IROL. (1989) 70:3501.Perhaps, can use liposome with recombinant expression vector and baculovirus transfection insect cell.For example referring to people such as Liebman, BIOTECHNIQUES (1999) 26 (1): 36; People such as Graves, BIOCHEMISTRY (1998) 37:6050; People such as Nomura, J.BIOL.C_HEM. (1998) 273 (22): 13570; People such as Schmidt, P_ROTEINE_{XPRESSION AND}P_URIFICATION(1998) 12:323; People such as Siffert, N_ATUREG_ENETICS(1998) 18:45; T_ILKINSDeng the people, C_ELLB_IOLOGY: A L_ABORATORYH_ANDBOOK145-154 (1998); People such as Cai, P_ROTEINE_{XPRESSION AND}P_URIFICATION(1997) 10:263; People such as Dolphin, N_ATUREG_ENETICS(1997) 17:491; People such as Kost, G_ENE(1997) 190:139; People such as Jakobsson, J.B_IOL.C_HEM. (1996) 271:22203; People such as Rowles, J.B_IOL.C_HEM. (1996) 271 (37): 22376; People such as Reversey, J.B_IOL.C_HEM. (1996) 271 (39): 23607-10; People such as Stanley, J.B_IOL.C_HEM. (1995) 270:4121; People such as Sisk, J.V_IROL. (1994) 68 (2): 766; With people such as Peng, B_IOT_ECHNIQUES(1993) 14.2:274.Commercially available liposome for example comprise Cellfectin  and Lipofectin  (Invitrogen, Corp., Carlsbad, CA).In addition, can use calcium phosphate transfection.Referring to TROTTER and WOOD, 39 M_{ETHODS IN}M_OLECULARB_IOLOGY(1995); Kitts, NAR (1990) 18 (19): 5667; With Mann and King, J.G_EN.V_IROL. (1989) 70:3501.

Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter becomes any dna sequence dna of mRNA for transcribing (3 ') in conjunction with shaft-like viral rna polymerase and start code sequence (for example structure gene) downstream.Promotor has transcription initiation region, and it places near the 5 ' end of encoding sequence usually.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.Bacilliform virus promoter also can have the second area that is called as enhanser, if it exists usually away from structure gene.In addition, express and can be modulated or composition.

Provide the promoter sequence that is particularly useful in the structure gene of the later stage of infectious cycle through transcribing in a large number.Example comprise the gene that is derived from the viral polyhedrin of coding sequence (people such as FRIESEN, The Regulation of Baculovirus GeneExpression is in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986);EP 0 127839 and 0 155 476) and coding p10 proteic gene (people such as Vlak, J.G_EN.V_IROL. (1988) 69:765).

The rhabdovirus expression vector that newly forms is packaged into infectious recombinant baculovirus and subsequently can the known technology purifying growth of those skilled in the art bacterial plaque.Referring to people such as Miller, BiOESSAYS (1989) 4:91; SUMMERS and SMITH, T_EXASA_GRICULTURALE_XPERIMENTS_TATIONB_ULLETINNo.1555 (1987).

Developed the recombination rhabdovirus expression vector that is used for infecting into several insect cells.For example, developed the recombinant baculovirus that is particularly useful for Aedes aegypti (ATCC numbers CCL-125), silkworm (ATCC numbers CRL-8910), drosophila melanogaster (ATCC numbering 1963), fall army worm and cabbage looper.Referring to WO 89/046,699; Wright, NATURE (1986) 321:718; People J.V such as Carbonell_IROL. (1985) 56:153; People M such as Smith_OL.C_ELL.B_IOL. (1983) 3:2156.Usually referring to people IN VITRO C such as Fraser_ELL.D_EV.B_IOL. (1989) 25:225.Clearer and more definite, the clone that is used for the rhabdovirus expression vector system generally includes (but being not limited to) Sf9 (fall army worm) (ATCC numbers CRL-1711), Sf21 (fall army worm) (Invitrogen Corp., catalog number (Cat.No.) 11497-013 (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five^TMBTI-TN-5B1-4 (cabbage looper).

The cell and the substratum that are used for and amalgamation and expression heterologous polypeptide direct in baculovirus/expression can be buied by the market, and cell culture technology is normally known to the those skilled in the art.

Intestinal bacteria and other prokaryotic organism bacterial expression technology are well-known in affiliated field.Can obtain to be used for the variety carrier of host bacterium.Described carrier can be the low or high multi-copy vector of single copy.Carrier can be used for the clone and/or expresses.In view of about the competent document of carrier, variety carrier buy and even the handbook of carrier and estriction map and feature is described, need not to set off a discussion herein.Well-known as institute, described carrier generally includes the mark that allows selection, and described mark can provide cytotoxicity medicament resistance, prototroph or immunity.Often have a plurality of marks, it provides different characteristics.

The bacterium promotor is for being transcribed into any dna sequence dna of mRNA in conjunction with bacteria RNA polysaccharase and start code sequence (for example structure gene) downstream (3 ').Promotor will have transcription initiation region, and it places near the 5 ' end of encoding sequence usually.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.The bacterium promotor also can have the second area that is called operon, and what it can be overlapped in the synthetic beginning of RNA adjoins the RNA polymerase binding site.Since gene inhibition albumen can in conjunction with operon and thereby suppress transcribing of specific gene, so operon allows transcribing of negative regulation (can inducing).The constructive expression can be taken place under the situation that exists less than the negative regulatory element such as operon.In addition, just regulating and control and to realize by gene activation albumen binding sequence,, be usually located near RNA polymerase binding sequence (5 ') so if described sequence exists.The example of gene activation albumen is meta-bolites activator (CAP), its help in the initial intestinal bacteria (E.coli) the lac operon transcribe (people such as Raibaud, A_NNU.R_EV.G_ENET. (1984) 18:173).Thereby regulating and expressing can be forward or negative sense, strengthens thus or reduces and transcribe.

The sequence of coding metabolic pathway enzyme especially provides the promoter sequence of usefulness.Example comprises the promoter sequence that is derived from the carbohydrate metabolism enzyme, such as semi-lactosi, lactose (lac) (people such as Chang, NATURE (1977) 198:1056) and maltose.Additional examples comprises the promoter sequence that is derived from biosynthetic enzyme, such as tryptophane (trp) (people such as Goeddel, Nuc.A_CIDSR_ES. (1980) 8:4057; People such as Yelverton, N_UCL.A_CIDSRES. (1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; The open case 036 776 of European patent and 121 No. 775, it is incorporated herein by reference).Beta galactosidase enzyme (bla) promoter systems (Weissmann (1981) " The cloning of interferon and othermistakes. " In Interferon 3 (Ed.I.Gresser)), phage PL (people such as Shimatake, N_ATURE(1981) 292:128) and T5 (United States Patent (USP) the 4th, 689, No. 406, it is incorporated herein by reference) promoter systems also provide the promoter sequence of usefulness.The preferred method of the present invention utilization is induced hGH such as the strong promoter of T7 promotor with high level.The example of described carrier is well-known in affiliated field and comprises available from the pET29 of Novagen series and be described in pPOP carrier among the WO99/05297 (it is incorporated herein by reference).Described expression system produces high-load hGH polypeptide and can not damage host cell viablity or growth parameter(s) in the host.

In addition, also play the bacterium promotor at the non-existent synthetic promoter of occurring in nature.For example, the transcriptional activating sequence of bacterium or phage promoter can be engaged in the operon sequence of another bacterium or phage promoter, thereby produces synthetic hybrid promoter (United States Patent (USP) the 4th, 551, No. 433, it is incorporated herein by reference).For example, the tac promotor is the heterozygosis trp-lac promotor that comprises trp promotor and lac operon sequence, and it is regulated and control [people such as Amann, G by the lac repressor_ENE(1983) 25:167; People such as de Boer, P_ROC.N_ATL.A_CAD.S_CI. (1983) 80:21].In addition, the bacterium promotor can comprise the promotor that has in conjunction with the non-bacterial origin of the natural generation of bacteria RNA polysaccharase and initial ability of transcribing.But the promotor of the non-bacterial origin of natural generation also coupling in compatible RNA polymerase in prokaryotic organism, to produce the high level expression of some genes.Phage t7 RNA polymerase/promoter systems is example (people such as Studier, J.MOL.BIOL. (1986) 189:113 of coupling promoter systems; People such as Tabor, ProcNatl.Acad.Sci. (1985) 82:107).In addition, hybrid promoter also can comprise phage promoter and intestinal bacteria operon zone (No. the 267 851, the open case of European patent).

Except the function on subsequence, effectively ribosome bind site also is applicable to the expression of alien gene in prokaryotic organism.In intestinal bacteria, ribosome bind site is called Shine-Dalgarno (SD) sequence and the length that comprises initiator codon (ATG) and be positioned at upstream from start codon 3-11 Nucleotide place is the sequence (people NATURE (1975) 254:34 such as Shine) of 3-9 Nucleotide.Think that the SD sequence is by pairing between the 3 ' end of SD sequence and intestinal bacteria 16S rRNA being promoted mRNA be incorporated into rrna (people such as Steitz, " Genetic signals and nucleotidesequences in messenger RNA ", In Biological Regulation and Development:Gene Expression (Ed.R.F.Goldberger, 1979)).In order to express eukaryotic gene and prokaryotic gene (people such as Sambrook with weak ribosome bind site, " Expression of cloned genes in Escherichia coli ", Molecular Cloning:ALaboratory Manual, 1989).

Term " host bacterium " or " bacterial host cell " mean the bacterium that can be used as or be used as the receptor of recombinant vectors or other transfer DNA.Described term comprises the offspring of the primitive bacteria host cell of transfection.Should be appreciated that because chance or the sudden change had a mind to, the offspring of single parental cell can on the form or with original parent complementary genome or total DNA in full accord.Being characterized as offspring such as the parental cell that fully is similar to the parent of the correlation properties of the nucleotide sequence that has coding hGH polypeptide is included among the offspring that this definition means.

The suitable host bacterium that selection is used to express the hGH polypeptide is well-known to those of ordinary skill in the field.When selecting the host bacterium that is used to express, appropriate host can comprise that displaying especially has the host that good inclusion body forms ability, low proteolytic activity and general health.Host bacterium can obtain from multiple source usually, it includes, but is not limited to inferior university's (Berkeley branch school) biophysics of Gary welfare and medicophysics is bacterium heredity reserve center (Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA)) and U.S. typical case culture collecting center (" ATCC ") (Manassas, VA).The bacterium (for example BL21) that is derived from the bacterium (for example W3110) of K bacterial strain or is derived from the B bacterial strain is used in the fermentation of industry/medicine usually.These bacterial strains are especially suitable, because its growth parameter(s) is very well-known and healthy.In addition, these bacterial strains are avirulence, based on safety and environment reason, its commercial be important.In an embodiment of the inventive method, escherichia coli host is the bacterial strain of BL21.In another embodiment of the inventive method, escherichia coli host is the bacterial strain that includes, but is not limited to the removal proteolytic enzyme of OMP-and LON-.In another embodiment of method of the present invention, described host cell bacterial strain is the species of pseudomonas, and it includes, but is not limited to Pseudomonas fluorescens, Pseudomonas aeruginosa and pseudomonas putida.The Pseudomonas fluorescens mutation 1 of called after bacterial strain MB 101 can be used as host strain and is used for the treatment of protein production method (Midland, MI purchase in World Wide Web dow.com) by The Dow Chemical Company.The United States Patent (USP) the 4th, 755,465 and 4,859 that is incorporated herein by reference is described the purposes of the host cell that pseudomonad strain produces as hGH for No. 600.

In case set up recombinant host cell bacterial strain (be about to express construct body has been introduced host cell and separation has the host cell that body is constructed in suitable expression), then cultivated described recombinant host cell bacterial strain being suitable for producing under the condition of hGH polypeptide.As apparent to the those skilled in the art, the method for cultivating described recombinant host cell bacterial strain depends on character and the host cell itself that body is constructed in used expression.The well-known method in field was cultivated under the recombinant host bacterial strain used usually.Recombinant host cell is incubated at the assimilable source that contains carbon, nitrogen and inorganic salt usually and contains VITAMIN, amino acid, somatomedin and other well-known albumen in affiliated field according to circumstances and cultivates in the liquid nutrient medium of additive.The liquid nutrient medium that is used to cultivate host cell can contain microbiotic and antifongin according to circumstances to prevent undesirable microorganism growth and/or to include, but is not limited to antibiotic compound to select to contain the host cell of described expression vector.

Recombinant host cell mode is in batches or continuously cultivated, simultaneously with mode collecting cell in batches or continuously (the hGH polypeptide accumulates under the situation in the cell therein) or collect culture supernatant.For being produced in the prokaryotic host cell preferred batch culture and cell harvesting.

HGH polypeptide of the present invention is purifying after being expressed in recombination system usually.Described hGH polypeptide can be by known method purifying from host cell in the field under multiple.Usually result from hGH polypeptide solubilizing poorly or soluble (with the form of inclusion body) in the bacterial host cell.In one embodiment of the invention, aminoacid replacement can be easy to form in the hGH polypeptide, and it is selected for increasing the described proteic deliquescent purpose of known method reorganization generation in method disclosed herein and the affiliated field of utilizing.Under soluble proteic situation, described albumen can be from the host cell lysate by centrifugal collection or can be further subsequently with cell homogenates.Under the proteic situation of bad dissolved, can add the compound that includes, but is not limited to polymine (PEI) and precipitate to impel the part soluble protein.The sedimentary albumen of institute subsequently can be expediently by centrifugal collection.The cleavable or the recombinant host cell that homogenizes discharge inclusion body to use the well-known method of multiple those of ordinary skill in the field in described cell.Host cell destroy or homogenize can use include, but is not limited to enzymatic lysis, ultrasonication, Du Ensi homogenizes or high pressure discharges the well-known technology of cracked and carries out.In an embodiment of method of the present invention, use high pressure release tech cracking e. coli host cell to discharge the inclusion body of hGH polypeptide.The productive rate of having found the soluble hGH polypeptide of inclusion body form can only once increase e. coli host cell by homogenizer by utilizing.When handling the inclusion body of hGH polypeptide, for such as dissolving, mechanical shearing or proteolyzing, minimizing and repeating the time of homogenizing is favourable not losing with maximization inclusion body productive rate.

Under can using subsequently in the field any in the known multiple suitable solubilizing agent make soluble or the sedimentary hGH polypeptide dissolving of institute.Preferably make the dissolving of hGH polypeptide with urea or Guanidinium hydrochloride.The volume of dissolved hGH polypeptide-BP is minimized, so that the batch size that is easy to manage that can be easy to use produces big batch.Can be important in the large-scale commercial applications environment of batch growth of this factor volume that recombinant host can thousands of liters therein.In addition, when in the large-scale commercial applications environment, making the hGH polypeptide,, if possible, then should avoid to damage the harsh chemicals of machinery and container or protein product itself in particular for human medicinal use.Show in the method for the invention and can use gentle denaturing agent urea to replace causticity denaturing agent guanidine hydrochloride dissolution hGH polypeptide inclusion body.Use urea significantly to reduce the risk that used stainless steel equipment in the manufacturing of hGH polypeptide and purge process is damaged, simultaneously effective dissolving hGH polypeptide inclusion body.

When the hGH polypeptide is produced as fusion rotein, preferably remove fusion sequence.Can be by enzymatic or chemical cracking, preferably finish the removal of fusion sequence by enzymatic lysis.Enzymatic removes fusion sequence and can use the well-known method of those skilled in the art to finish.The selection that is used to remove the enzyme of fusion sequence will be determined by the characteristic of syzygy, and reaction conditions determined by the selection of enzyme, as apparent to the those skilled in the art.Preferably with well-known method from through cracked fusion sequence purifying through cracked hGH polypeptide.These methods are by fusion sequence and hGH polypeptide itself and character decision, as apparent to the those skilled in the art.The method that is used for purifying can include, but is not limited to size exclusion chromatogram, hydrophobic interaction chromatograph, ion-exchange chromatography or dialysis or its any combination.

Preferably also the described hGH polypeptide of purifying from protein solution, to remove DNA.DNA can remove by known appropriate method in the field under any, such as precipitation or ion-exchange chromatography, but preferably by using the nucleic acid precipitation agent precipitation such as, but not limited to protamine sulfate to remove.Described hGH polypeptide can use and include, but is not limited to the well-known method of centrifugal or filtering standard and separate from deposit D NA.Remove host's nucleic acid molecule therein the hGH polypeptide will be used for the treatment of in the human environment for important factor and method of the present invention and reduce host cell DNA to pharmaceutically acceptable level.

The method that also can be used for small-scale or large scale fermentation in protein expression, it includes, but is not limited to fermentor tank, vibration bottle, fluidized bed bio reactor, hollow fiber bio-reactor, rolling bottle culture systems and steel basin bioreactor system.These methods various can be in batches, fed-batch or continuous mode method are carried out.

Standard method under human hGH polypeptide of the present invention can use usually in the field is reclaimed.For example, can be centrifugal or filter substratum or cell lysates to remove cell debris.Can with supernatant concentration or be diluted to the volume of wanting or filter the preparation that is used to be further purified with adjusting to the suitable buffer thoroughly.Be further purified hGH polypeptide of the present invention and comprise deacylated tRNA amine and the clipped form that separates the hGH polypeptide variants from complete form.

Any following exemplary program can be used for purifying hGH polypeptide of the present invention: affinity chromatography, negatively charged ion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE), silica gel chromatography, reversed-phase HPLC, gel-filtration (use includes, but is not limited to SEPHADEX G-75), hydrophobic interaction chromatograph, the size exclusion chromatogram, immobilized metal ion afinity chromatography, ultrafiltration/saturating filter, ethanol sedimentation, ammonium sulfate precipitation, chromatofocusing, displcement chromatography, electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation property), dissolubility difference (including, but is not limited to ammonium sulfate precipitation), SDS-PAGE or extraction.

The standard program of can be known according to the those skilled in the art and using is with protein part of the present invention ground or be purified to homogeneous substantially, described albumen include, but is not limited to comprise alpha-non-natural amino acid albumen, the anti-proteic antibody that comprises alpha-non-natural amino acid, comprise the proteic of alpha-non-natural amino acid in conjunction with the thing etc. of arranging in pairs or groups.Therefore, can under in the field any in the well-known several different methods reclaim and purifying polypeptide of the present invention, described method includes, but is not limited to ammonium sulfate or ethanol sedimentation, acid or alkali extraction, tubing string chromatogram, affine tubing string chromatogram, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, hydroxyapatite chromatography, lectin chromatogram, gel electrophoresis or the like.In case of necessity, can correctly be folded into use protein folding step in several albumen in generation.High performance liquid chromatography (HPLC), affinity chromatography or other appropriate method can be used for wherein wishing highly purified final purification step.In one embodiment, the antibody of the anti-alpha-non-natural amino acid that the is produced albumen of alpha-non-natural amino acid (or comprise) includes, but is not limited to be used for comprising based on affinity purification the albumen of one or more alpha-non-natural amino acids as purified reagent.In case of necessity, after partially or completely being purified to homogeneous, described polypeptide is used for multiple effectiveness according to circumstances, includes, but is not limited to as the immunogen of examining and determine component, therapeutical agent, preventive, diagnostic reagent, research reagent and/or producing as antibody.

Except other reference that this paper mentions, multiple purifying/protein folding method is well-known in affiliated field, and it includes, but is not limited to the described method of following document: R.Scopes,Protein PurificatioN, Springer-Verlag, N.Y. (1982); Deutscher,Methods in EnzvmoloRy Vol.182:Guide to Protein Purification, Academic Press, Inc.N.Y. (1990); Sandana, (1997)Bioseparation of Proteins, AcademicPress, Inc.; People such as Bollag, (1996)Protein Methods, 2nd Edition Wiley-Liss, NY; Walker, (1996)The Protein Protocols HandbookHumana Press, NJ, Harris and Angal, (1990)ProteinPurification Applications:A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal,Protein Purification Methods:A Practical ApproachIRL Press at Oxford, Oxford, England; Scopes, (1993)Protein Purification:Principles and Practice 3rd EditionSpringer Verlag, NY; Janson and Ryden, (1998)Protein Purification:Principles, HighResolution Methods and Applications, Second EditionWiley-VCH, NY; And Walker (1998),Protein Protocols on CD-ROMHumana Press, NJ; And the reference of wherein being quoted.

In eukaryotic host cell or non-eukaryotic host cell, produce have alpha-non-natural amino acid an advantage of the albumen of paying close attention to or polypeptide be that described albumen or polypeptide will be folded into its native conformation usually.But, in certain embodiments of the present invention, it will be understood by one of ordinary skill in the art that albumen can have the conformation of the conformation of wanting that is different from related polypeptide behind synthetic, expression and/or purifying.In one aspect of the invention, with expressed protein denaturation and renaturation subsequently.Under it utilizes in the field known method finish, described method include, but is not limited to by add chaperone to associated protein or polypeptide, by described albumen being dissolved in the chaotropic agent such as Guanidinium hydrochloride, using protein disulfide bond isomerase etc.

Generally speaking, wish that sometimes the sex change or the expressed polypeptide that reduces also make described polypeptide be folded into preferred conformation more then.For example, can add guanidine, urea, DTT, DTE and/or chaperone to relevant translation product.The method of reduction, sex change and recombinant protein is well-known (referring to people such as above reference and Debinski, (1993) to the those skilled in the artJ.Biol.Chem., 268:14065-14070; Kreitman and Pastan (1993)Bioconiug.Chem..4:581-585; With people such as Buchner, (1992)Anal.Biochem.,205:263-270).For example people such as Debinski has described sex change and the reduction of inclusion body albumen in guanidine-DTE.Albumen can be folding again in containing the arginic potential buffer solution of (including, but is not limited to) oxidized glutathione and L-.Folding again reagent can flow into or otherwise enter contacting with one or more polypeptide or other expression product, or vice versa.

Produce at eucaryon under the situation of hGH polypeptide, so the hGH polypeptide possible errors that produces folds and thereby lacks biological activity or have the biological activity of reduction.Described proteic biological activity can be recovered by " folding again ".Generally speaking, dissolve (wherein said hGH is also for insoluble), separate folding and the reducing polypeptide chain folds the hGH polypeptide of false folding again by using one or more chaotropic agents (for example urea and/or guanidine) for example and can reducing the reductive agent (for example dithiothreitol (DTT), DTT or 2 mercapto ethanol) of disulfide linkage.Under the chaotropic agent of intermediate concentration, add oxygenant (for example oxygen, halfcystine or cystamine) subsequently, make to form disulfide linkage again.Under can using in the field known standard method make the hGH polypeptide folding again, such as being described in United States Patent (USP) the 4th, 511,502,4,511,503 and 4,512, the method in No. 922 (it is incorporated herein by reference).The hGH polypeptide also can be folded to form heterodimer or heteropolymer altogether with other albumen.After folding again or folding altogether, preferably the hGH polypeptide is further purified.

General purification process can any suitable order carries out arbitrary step in the multiple separating step to the cell lysates that comprises the hGH polypeptide or any hGH polypeptide mixture of deriving from any separating step, and it includes, but is not limited to affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatograph, gel filtration chromatography, high performance liquid chromatography (" HPLC "), reversed-phase HPLC (" RP-HPLC "), expanded bed adsorption or its any combination and/or circulation.

The equipment and other essential material that are used to carry out technology described herein can be buied.Pump, run tank, watch-dog, registering instrument and total system can be available from for example Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway, NJ).The chromatographic material that includes, but is not limited to exchange group material, medium and damping fluid also can be available from described company.

Can use such as the Special Equipment of pump is quicker such as the balance in the washing and the tubing string chromatographic process as herein described of elution and other step and to finish.Commercially available pump include, but is not limited to HILOAD  pump P-50, peristaltic pump P-1, pump P-901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).

The example of run tank comprise RediFrac run tank, FRAC-100 and FRAC-200 run tank and SUPERFRAC  run tank (Amersham Biosciences, Piscataway, NJ).Mixing tank also can be used for forming pH value and linear concentration gradient.Commercially available mixing tank comprise gradient mixer GM-1 and straight-line type mixing tank (Amersham Biosciences, Piscataway, NJ).

Chromatographic process can be used any commercially available watch-dog monitoring.Described watch-dog can be used for collecting the information as UV, pH and electroconductibility.The example of detector comprise watch-dog UV-1, UVICORD  S II, watch-dog UV-M II, watch-dog UV-900, watch-dog UPC-900, watch-dog pH/C-900 and electroconductibility watch-dog (Amersham Biosciences, Piscataway, NJ).In fact, total system can be buied, and it comprises Amersham Biosciences (Piscataway, various AKTA  system NJ).

In one embodiment of the invention, for example, by at first in urea with the sex change of gained purifying hGH polypeptide, dilute in the TRIS damping fluid under containing the suitable pH value of reductive agent (such as DTT) subsequently, can make hGH polypeptide reduction and sex change.In another embodiment, the hGH polypeptide in about 2M sex change to the concentration range of about 9M, dilutes under the pH value of about 5.0 to about 8.0 scope in the TRIS damping fluid in urea then.Can cultivate the folding again mixture of this embodiment then.In one embodiment, folding again mixture was at room temperature cultivated four hours to 24 hours.Can will further separate and purifying with the hGH polypeptide mixture of sex change then through reduction.

PH value as described herein, as can before carrying out any separating step subsequently, to regulate a hGH polypeptide mixture.In addition, can use the concentrated hGH polypeptide mixture of known technology or its any mixture subsequently in the affiliated field.In addition, the elution buffer-exchanged that can use the well-known technology of those of ordinary skill in the field will comprise a described hGH polypeptide mixture and any mixture subsequently thereof is the damping fluid that is suitable for following separating step.

Ion-exchange chromatography can carry out ion-exchange chromatography to a hGH polypeptide mixture in one embodiment and as optional additional step.Usually referring to ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES ANDMETHODS (catalog number (Cat.No.) 18-1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion-exchange tubing string comprise HITRAP , HIPREP  and HILOAD  tubing string (Amersham Biosciences, Piscataway, NJ).Described tubing string utilizes strong anion exchanger, such as Q SEPHAROSE  Fast Flow, Q SEPHAROSE  High Performance and Q SEPHAROSE  XL; Strong cation exchanger is such as SP SEPHAROSE  HighPerformance, SP SEPHAROSE  Fast Flow and SP SEPHAROSE  XL; Weak anion exchanger is such as DEAE SEPHAROSE  Fast Flow; And weak cation exchanger, such as CM SEPHAROSE  FastFlow (Amersham Biosciences, Piscataway, NJ).Can carry out cationic exchange tubing string chromatogram to separate the hGH polypeptide of purifying substantially to the hGH polypeptide in any stage of purge process.Can use any suitable cationic exchange matrix to carry out the cation-exchange chromatography step.The cationic exchange matrix that is suitable for includes, but is not limited to fibering, porousness, imporosity, microgranular, beadization and cross-linked cationic exchange group material.These cationic exchange substrate materials include, but is not limited to the mixture of Mierocrystalline cellulose, agarose, dextran, polyacrylic ester, polyethylene, polystyrene, silicon-dioxide, polyethers and any aforementioned substances.After the hGH polypeptide being adsorbed on the described cationic exchange matrix, by making damping fluid that matrix contact has sufficiently high pH value and an ionic strength so that the hGH polypeptide is replaced from described matrix, but the elution hGH polypeptide of purifying substantially.Be used for high pH elution substantially the suitable buffer of the hGH polypeptide of purifying include, but is not limited to concentration range and be at least about 5mM at least about Citrate trianion, phosphoric acid salt, formate, acetate, HEPES and the MES damping fluid of 100mM.

Reverse-phase chromatography can carry out RP-HPLC with purifying protein according to the known suitable scheme of those of ordinary skill in the field.For example referring to people ANAL BlOCHEM. (1982) 124:217-230 (1982) such as Pearson; People J.CHROM. (1983) 268:112-119 such as Rivier; People J.CHROM. (1986) 359:391-402 such as Kunitani.RP-HPLC can carry out on the hGH polypeptide to separate the hGH polypeptide of purifying substantially.In this, can use alkyl functional base with multiple length through silicon-dioxide deutero-resin, described length includes, but is not limited at least about C3 at least about C30, at least about C3 at least about C20 and at least about C3 at least about C18.Perhaps, can use fluoropolymer resin.For example, can use TosoHaas Amberchrome CG1000sd resin, it is a styrenic polymer resins.Also can use cyano group and polymer resin with multiple alkyl chain length.In addition, can be such as alcoholic acid solvent wash RP-HPLC tubing string.Can use the suitable elution damping fluid that contains agent of ion antithesis and organic regulator (such as methyl alcohol, Virahol, tetrahydrofuran (THF), acetonitrile or ethanol) from RP-HPLC tubing string elution hGH polypeptide.The most frequently used ion antithesis agent includes, but is not limited to acetate, formic acid, perchloric acid, tetrafluoro acetate, hexafluoro butyric acid, triethylamine, acetate tetramethylammonium, acetate tetrabutylammonium, acetate Tetrylammonium.Can use a kind of gradient and isocratic condition with more than one to carry out elution, the preferred gradient condition is to reduce disengaging time and to reduce peak width simultaneously.Other method comprises uses two gradients with different solvents concentration range.The example that is used for the suitable elution damping fluid of this paper can include, but is not limited to ammonium acetate and acetonitrile solution.

The hydrophobic interaction chromatograph purification technique can carry out hydrophobic interaction chromatograph (HIC) to the hGH polypeptide.Usually referring to H_YDROPHOBICI_NTERACTIONC_{HROMATOGRAPHY}H_ANDBOOK: P_{RINCIPLES AND}M_ETHODS(it incorporates this paper by reference into for catalog number (Cat.No.) 18-1020-90, Amersham Biosciences (Piscataway, NJ)).Appropriate H IC matrix can include, but is not limited to the matrix through the alkyl or aryl replacement, such as the matrix that comprises agarose, Sepharose, sepharose, Mierocrystalline cellulose, silicon-dioxide, dextran, polystyrene, poly-(methacrylic ester) matrix and mixed mode resin that replaces through butyl, hexyl, octyl group or phenyl, poly-(methacrylic ester) matrix that it includes, but is not limited to the polyvinylamine resin or replaces through butyl or phenyl.The commercially available source of hydrophobic interaction tubing string stratographic include, but is not limited to HITRAP , HIPREP  and HILOAD  tubing string (Amersham Biosciences, Piscataway, NJ).In brief, before charging, can use the known standard buffer solution balance of those of ordinary skill in the field HIC tubing string, described standard buffer solution such as acetate/sodium chloride solution or contain the HEPES of ammonium sulfate.After loading the hGH polypeptide, can use standard buffer solution and condition washing tubing string removing unwanted material subsequently, but the hGH polypeptide is remained on the HIC tubing string.The available about 3 standard buffer solution elution hGH polypeptide to about 10 tubing string volumes, described damping fluid is such as especially for containing the HEPES or the acetate/sodium-chlor damping fluid of EDTA and the ammonium sulfate concentrations lower than level pad.For example using, potassiumphosphate gradient reduction linear salt gradient can be used for elution hGH molecule.For example, can concentrate eluant by filtration method then such as saturating filter or ultrafiltration.Can use filter and remove the salt that is used for elution hGH polypeptide.

Other purification technique can use example gel to filter (G to a hGH polypeptide mixture or its any mixture subsequently_ELF_ILTRATION: P_{RINCIPLES AND}M_ETHODS(catalog number (Cat.No.) 18-1022-18, AmershamBiosciences, Piscataway, NJ), it is incorporated herein by reference), another separating step of HPLC, expanded bed adsorption, ultrafiltration, saturating filter, freeze-drying or the like, removing any excessive salt, even and replace described damping fluid with suitable buffer and allocate final medicament production to be used for subsequently separating step.Can use the known technology of those of ordinary skill in the field in each step as herein described, to monitor and comprise the productive rate of the hGH polypeptide of the hGH polypeptide of purifying substantially.These technology are assessed the productive rate of the hGH polypeptide of purifying substantially after also being used in last separating step.For example, can use such as cyano group RP-HPLC, C₁₈Any of several anti-phase high pressure liquid chromatography tubing strings with multiple alkyl chain length of RP-HPLC and cationic exchange HPLC and gel-filtration HPLC monitored the productive rate of hGH polypeptide.

Can use such as the standard technique of SDS-PAGE or by using Western blot and ELISA calibrating measuring h GH polypeptide to measure purity.For example, can produce the anti-proteic polyclonal antibody that separates from negative control yeast fermentation and cationic exchange recovery.Also can use described antibody to survey the existence of contaminative host cell proteins.

RP-HPLC material Vydac C4 (Vydac) is made up of the silica gel particle that the surface has the C4-alkyl chain.It is different that hGH polypeptide and albumen impurity are separated the intensity that is based on hydrophobic interaction.Carry out elution with the acetonitrile gradient in rare trifluoroacetic acid.Use stainless-steel tubing pillar (the Vydac C4 silica gel that filling is 2.8 to 3.2 liters) being prepared property HPLC.Come acidifying hydroxylapatite Ultrogel elutant and be loaded on the Vydac C4 tubing string by adding trifluoroacetic acid.For washing and elution, be used in the acetonitrile gradient in rare trifluoroacetic acid.Collect molten from part and neutralizing with phosphate buffered saline buffer immediately.The hGH polypeptide that compiles in the IPC restriction is molten from part.

DEAE sepharose (Pharmacia) material is made up of diethylin ethyl (the DEAE)-group that is covalently bonded in the sepharose surface of beads.The hGH polypeptide is by ionic interaction mediation with combining of DEAE group.Acetonitrile and trifluoroacetic acid are not detained by tubing string.After these materials wash out, by under low pH value, removing trace impurity with acetate buffer washing tubing string.Subsequently with tubing string with neutral phosphonic phthalate buffer washing and with the damping fluid elution hGH polypeptide of ionic strength with increase.Tubing string is loaded with DEAE Sepharose fast flow.The adjustable pipe column volume loads with the scope of 3-10 milligram hGH polypeptide/milliliter gel to guarantee the hGH polypeptide.Water and level pad (sodium phosphate/potassium) washing tubing string.The molten of the HPLC elutant that loading compiles washed with level pad from part and with tubing string.Then tubing string is washed with lavation buffer solution (sodium acetate buffer), wash with level pad subsequently.Then, the hGH polypeptide is collected from part with Dan Rong from the tubing string elution and according to main elution curve with elution damping fluid (sodium-chlor, sodium phosphate/potassium).The elutant of DEAE sepharose tubing string is adjusted to the appointment electric conductivity.Be aseptically filled into the gained drug substance in Teflon (Teflon) bottle and be stored in-70 ℃.

Can use several different methods and program to assess proteic productive rate of the hGH that contains one or more non-naturally encoded amino acids and purity, described method includes, but is not limited to Bradford calibrating, SDS-PAGE, silver and dyes SDS-PAGE, Ku Masi dyeing SDS-PAGE, mass spectrum (including but not limited to MALDI-TOF) and known other method that is used for profiling protein of those skilled in the art.

VIII. the expression in alternative system

Brought out in non-recombinant hosts cell, sudden change and become host cell or in cell free system, use some strategies alpha-non-natural amino acid is introduced in the albumen.Described system also is applicable to and makes hGH polypeptide of the present invention.The amino acid whose derivatization with reactive side chain such as Lys, Cys and Tyr makes Methionin change N into²-ethanoyl-Methionin.Chemosynthesis also provides the direct method of incorporating alpha-non-natural amino acid into.In view of the enzymatic of nearest peptide fragment connects the development that is connected with native chemical, may make bigger albumen.For example referring to P.E.Dawson and S.B.H.Kent,Annu.Rev.Biochem., 69:923 (2000).Wherein will add the general external biological synthetic method that can support in the external extract of protein biology synthetic with the inhibition tRNA of want alpha-non-natural amino acid chemical acylation has been used for incorporating the albumen of multiple almost any size into surpassing 100 alpha-non-natural amino acid locus specificity ground.For example referring to V.W.Cornish, D.Mendel and P.G.Schultz,Angew.Chem.Int.Ed.Engl.1995,34:621 (1995); CJ.Noren, SJ.Anthony-Cahill, M.C.Griffith, P.G.Schultz, A general method for site-specific incorporation of unnaturalamino acids into proteins,Science244:182-188 (1989); And J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specific incorporation of a non-natural aminoacid mto a polypeptide, LAm.Chem.Soc.111:8013-8014 (1989).The functional group introduces and is used to study protein stability, protein folding, enzyme mechanism and signal transduction in the albumen widely.

Exploitation is called as in the body that selective pressure incorporates into method to utilize the crossability of wild-type synthetic enzyme.For example referring to N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber,FASEB J., 13:41 (1999).Grow in the minimal medium of the natural amino acid that contains limited concentration wherein supplying the specific natural amino acid whose associated metabolic of the cell auxotrophic strain that the path is closed, transcribing of target gene is suppressed simultaneously.In the steadily beginning in vegetative period, natural amino acid is depleted and replace with the alpha-non-natural amino acid analogue.Induce Recombinant Protein Expression can cause containing the protein accumulation of non-natural analogue.For example, use this strategy, with the neighbour, and P-fluoropnenylalanine incorporate in the albumen, and in the UV spectrum that can be easy to discern, represent two characteristic acromions, for example referring to C.Minks, R.Huber, L.Moroder and N.Budisa,Anal.Biochem., 284:29 (2000); In phage T4 N,O-Diacetylmuramidase, used fluoroform methyllanthionine displacement methionine(Met) to study the interaction of itself and oligochitosan ligand by 19F NMR, for example referring to H.Duewel, E.Daub, V.Robinson and J.F.Honek,Biochemistry, 36:3404 (1997); And incorporated the trifluoro leucine into the replacement leucine, thereby caused the thermostability of leucine zipper protein and chemical stability to increase.For example referring to Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew.Chem.Int.Ed.Engl., 40:1494 (2001).In addition, selenomethionine and telluro methionine(Met) being incorporated into various recombinant proteins is beneficial to resolve in the X-ray crystallography mutually.For example referring to W.A.Hendrickson, J.R.Horton and D.M.Lemaster,EMBO J., 9:1665 (1990); J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and M.Hatada,Nat.Struct.BioL1:283 (1994); N.Budisa, B.Steipe, P.Demange, C.Eckerskom, J.Kellermann and R.Huber,Eur.J.Biochem..230:788 (1995); And N.Budisa, W.Kambrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and R.Huber,J.Mol.Biol..270:616 (1997).Also incorporated methionine(Met) analogue effectively into, thereby allowed chemically other modified protein with alkene or alkynes functional group.For example referring to J.C.M.vanHest and D.A.Tirrell,FEBS Lett..428:68 (1998); J.C.M.van Hest, K.L.Kiick and D.A.Tirrell,J.Am.Chem.Soc, 122:1282 (2000); With K.L.Kiick and D.A.Tirrell, Tetrahedron, 56:9487 (2000); United States Patent (USP) the 6th, 586, No. 207; U.S. Patent Publication case 2002/0042097, it is incorporated herein by reference.

The success of this method depends on the identification of aminoacyl-tRNA synthetase to the alpha-non-natural amino acid analogue, and it generally requires highly selective to guarantee the fidelity of reproduction of protein translation.A kind of mode of expanding the category of this method is to relax the substrate specificity of aminoacyl-tRNA synthetase, and it is realized under the situation of limited quantity.For example, in intestinal bacteria phenylalanyl-tRNA synthetic enzyme (PheRS), replace Ala with Gly²⁹⁴Can increase the size of substrate binding pocket and cause tRNAPhe by fenclonine (p-Cl-Phe) acidylate.Referring to M.Ibba, P.Kast and H.Hennecke,Biochemistry, 33:7107 (1994).The e. coli strains that comprises this sudden change PheRS allow to incorporate into fenclonine and to bromophenyl alanine to replace phenylalanine.For example referring to M.Ibba and H.Hennecke,FEBS Lett..364:272 (1995); And N.Sharma, R.Furter, P.Kast and D.A.Tirrell,FEBS Lett., 467:37 (2000).Similarly, near the displaying of the point mutation Phel30Ser the amino acid binding site of intestinal bacteria tyrosyl-tRNA synthetic enzyme allows azatyrosine more effectively to incorporate into than tyrosine.Referring to F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura,J.Biol.Chem..275:40324 (2000).

Incorporating alpha-non-natural amino acid into proteic another strategy in the body is to modify the synthetic enzyme with check and correction mechanism.These synthetic enzyme can not be similar to the amino acid of homology natural amino acid and thereby activate it on the specification configuration.This mistake obtains in independent site proofreading and correct, and it goes acidylate to keep the fidelity of reproduction of protein translation from tRNA in the amino acid on the misloading.If the check and correction loss of activity of synthetic enzyme, then wrong activatory analog can be escaped editting function and be merged in.Used valyl-tRNA synthetic enzyme (ValRS) to prove this method recently.Referring to V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere,Science, 292:501 (2001).ValRS can use Cys, Thr or the wrong aminoacylation tRNAVal of aminobutyric acid ester (Abu); These non-homogeneous amino acid are subsequently by the edit field hydrolysis.After bringing out escherichia coli chromosome sudden change at random, be chosen in the mutant Escherichia coli strain that has sudden change in the edit field of ValRS.This editor's defective type ValRS loads tRNAVal mistakenly with Cys.Because be similar to Cys (in Abu Cys-the SH group is with-CH3 displacement) on the Abu space, when this mutant Escherichia coli strain is grown in the presence of Abu so the ValRS that suddenlys change also incorporates Abu into albumen.Mass spectroscopy shows that Xie Ansuan each Xie Ansuan position in former raw albumen of about 24% replaced by Abu.

Solid phase synthesis and semisynthesis also allow the synthetic multiple albumen that contains new amino acid.For example, reach the reference of wherein being quoted referring to the following discloses case, it is as follows: Crick, F.J.C., Barrett, L.Brenner, S.Watts-Tobin, R.General nature of the genetic code for proteins.Nature, 192:1227-1232 (1961); Hofmann, K., Bohn, H.Studies on polypeptides.XXXVI.The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptidefragment,J.Am Chem, 88 (24): 5914-5919 (1966); Kaiser, E.T.Synthetic approaches tobiologically active peptides and proteins including enyzmes,Ace Chem Res, 47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T.Peptide segment coupling catalyzed by thesemisynthetic enzyme thiosubtilisin, JAm Chem Soc, 3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease,Science, 256 (5054): 221-225 (1992); Chaiken, I.M.Semisynthetic peptides and proteins,CRC Crit Rev Biochem, 11 (3): 255-301 (1981); Offord, R.E.Protein engineering by chemical means?ProteinEng., 1 (3): 151-157 (1987); And Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A Designed Peptide Ligase forTotal Synthesis of Ribonuclease A with Unnatural Catalytic Residues,Science, 266 (5183): 243 (1994).

Used chemically modified will comprise the external introducing albumen of non-natural side chain of multiple cofactor, spin labeling and oligonucleotide.For example referring to Corey, D.R., Schultz, P.G.Generation of a hybrid sequence-specificsingle-stranded deoxyribonuclease,Science, 238 (4832): 1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymatic specificity, AnnuRevBiochem, 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S.Chemical mutation of enyzmeactive sites,Science, 226 (4674): 505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin,Biol.Chem, 243 (24): 6392-6401 (1968); Polgar, L.B., MX.Anew enzyme containing a synthetically formed acttve site.Thiol-subtilisin.J.Am Chem Soc, 3153-3154 (1966); And Pollack, S.J., Nakayama, G.Schultz, P.G.Introduction of nucleophilesand spectroscopicprobes into antibody combining sites,Science, 242 (4881): 1038～1040 (1988).

Perhaps, the biosynthetic means that has used applied chemistry to modify the ground aminoacyl-tRNA is incorporated several biophysics probe bodies into institute synthetic albumen outward.Reach the reference of wherein being quoted: Brunner referring to the following discloses case, J.NewPhotolabeling and crosslinking methods,Annu.Rev Biochem, 62:483-514 (1993); And Krieg, U.C., Walter, P., Hohnson, A.E.PhotocrosslinUng of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the sighal recognition particleProc.Natl.Acad.Sci, 83 (22): 8604-8608 (1986).

Proved to add by inhibition tRNA with the protein synthesis reaction that contains the gene programming of wanting the amber nonsense mutation to some extent before and incorporated the external locus specificity of alpha-non-natural amino acid ground into albumen chemical aminoacylation.Use these methods, people can use the auxotrophic strain of specific amino acids that multiple common 20 seed amino acids are replaced with the approximate construction homologue, for example fluorophenylalanine substituted benzene L-Ala.For example referring to Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G.Ageneral method for site-specific incorporation of unnatural amino acids intoproteins,Science, 244:182-188 (1989); People such as M.W.Nowak,Science268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specificIncorporation of a non-natural amino acid into a polypeptide,J.Am Chem Soc, 111:8013-8014 (1989); People such as N.Budisa,FASEB J.13:41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosynthetic method for introducing unnaturalamino acids site-specificaily into proteins.Methods in Enz., 301-336 (1992); And Mendel, D., Cornish, V.W.﹠amp; Schultz, P.G. Site-Directed Mutagenesis with an Expanded Genetic Code,Annu Rev Biophys.Biomol Struct.24,435-62 (1995).

For example, the inhibition tRNA of preparation identification terminator codon UAG is also with alpha-non-natural amino acid chemistry aminoacylation.Use conventional rite-directed mutagenesis to bring out and introduce terminator codon TAG at the related locus of protein gene.For example referring to Sayers, J.R., Schmidt, W.Eckstein, F.5 ', 3 ' Exonuclease in phosphoi ' othioate-basedolignoucleotide-directed mutagensis,Nucleic Acids Res, 16 (3): 791-802 (1988).When acidylate being suppressed tRNA and mutator gene and is combined in in-vitro transcription/translation system, to reply the UAG codon and incorporate alpha-non-natural amino acid into, it is created in specified location and contains described amino acid whose albumen.Use [³H]-experiment of Phe and use experimental results show that of alpha hydroxy acid only to want amino acid to be merged in and do not incorporate any other site in the albumen into by UAG codon appointed positions and this amino acid.For example, the same referring to people such as Noren, people such as Kobayashi, (2003) Nature StructuralBiology 10 (6): 425-432; And Ellman, J.A., Mendel, D., Schultz, P.G.Site-specificincorporation of novel backbone structures into proteins,Science, 255 (5041): 197-200 (1992).

Used the microinjection technology to incorporate alpha-non-natural amino acid into albumen.For example referring to M.W.Nowak, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester,Science, 268:439 (1995); And D.A.Dougherty,Curr.Opin.Chem.Biol., 4:645 (2000).Inject the RNA material of two kinds of external preparations simultaneously to xenopus leavis oocytes: a kind of mRNA and a kind of amber inhibition tRNA that has the coding target protein of UAG terminator codon in the related amino acid position through want alpha-non-natural amino acid aminoacylation.The body translation of ovocyte is inserting alpha-non-natural amino acid by the UAG appointed positions then.This method has allowed not to be subjected to usually the proteic body inner structure of the conformity membrane functional study of vivoexpression systemic effect.Example comprises incorporates fluorescence amino acid into tachykinin neurokinin-2 acceptor to come measuring distance by FRET (fluorescence resonance energy transfer), for example referring to G.Turcatti, and K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.CholletJ.Biol.Chem., 271:19991 (1996); Incorporate the residue that biotinylation amino acid exposes with surface in the identification ionic channel into, for example referring to J.P.Gallivan, H.A.Lester and D.A.Dougherty,Chem.Biol., 4:739 (1997); Use cage shape tyrosine analogue with the conformational change in the real-time monitoring ion passage, for example referring to J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619 (1998); Change the ionic channel skeleton to survey its door control mechanism with use α hydroxy-amino-acid.For example referring to P.M.England, Y.Zhang, D.A.Dougherty and H.A.Lester, Cell, 96:89 (1999); And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang,Nat.Neurosci.4:239 (2001).

The ability in the albumen directly alpha-non-natural amino acid incorporated in the body provides high mutain productive rate, technology simplification, in cell or may in Living Organism, study the possibility of mutain and the advantage of these mutains of use in the therapeutic type treatment.The alpha-non-natural amino acid that will have all size, acidity, nucleophilicity, hydrophobicity and other characteristic includes ability in the albumen can expand ability that we rationally and systematically handle protein structure dramatically to survey new albumen or the organism that protein function and generation have novel characteristics.Yet described method is difficult, because realize the interactional complicated character of the needed tRNA-synthetic enzyme of fidelity of reproduction of high level in protein translation.

Incorporate at locus specificity in the trial of right-F-Phe, anti-right-phenylalanyl-the tRNA synthetic enzyme is right to use the yeast amber to suppress tRNAPheCUA/ in the Phe auxotroph e. coli strains of F-Phe resistance.For example referring to R.Furter,Protein Sci..7:419 (1998).

Also may use acellular (external) translation system to obtain the expression of hGH polynucleotide of the present invention.Can comprise that therein mRNA in these systems as template (with in-vitro transcription and translation combination), is instructed external synthetic as template (external translation) or DNA by rrna.Had and significantly made great efforts to be used to develop the acellular albumen expression system.For example referring to Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering, 74:309～316 (2001); Kim, D.-M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology Progress, 16,385-390, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering, 66,180-188, (1999); And Patnaik, R. and J.R.Swartz, Biotechniques 24,862-868, (1998); United States Patent (USP) the 6th, 337, No. 191, No. the 2002/0081660th, U.S. Patent Publication case, WO 00/55353, WO 90/05785, it is incorporated herein by reference.The other method that can be used for expressing the hGH polypeptide that comprises non-naturally encoded amino acids comprises mRNA-peptide integration technology.For example referring to R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94:12297-12302 (1997); People such as A.Frankel, Chemistry ﹠amp; Biology 10:1043-1050 (2003).In this method, the mRNA template that will be connected in tetracycline on rrna is translated as peptide.If one or more tRNA molecules are modified, then alpha-non-natural amino acid also can be incorporated in the peptide.After in the end a mRNA codon was read, tetracycline was caught the C-terminal of described peptide.Have noticeable characteristic if find gained mRNA-peptide joiner in external calibrating, then its characteristic is easy to by the mRNA sequence open.By this way, people can screen the library of the hGH polypeptide that comprises one or more non-naturally encoded amino acids has the characteristic of being wanted with identification polypeptide.Recently, reported the synthetic peptide that replaces through non-naturally encoded amino acids of external rrna translation permission that utilizes the purifying component.For example referring to people such as Forster, Proc.Natl Acad.Sci. (USA) 100:6353 (2003).

IX. coupling is in the high polymer of hGH polypeptide

Can use composition as herein described, method, technology and strategy to rely and realize the various modifications to non-natural amino acid polypeptides as herein described.These modifications comprise to be incorporated another functional group on the alpha-non-natural amino acid component of polypeptide into, it includes, but is not limited to mark, dyestuff, polymkeric substance, water-soluble polymers, polyethyleneglycol derivative, the photocrosslinking agent, cytotoxic compound, medicine, affinity labelling, photoaffinity labeling, reactive compounds, resin, second albumen or polypeptide or polypeptide analog, antibody or antibody fragment, metal chelator, cofactor, lipid acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotides, inhibition Yeast Nucleic Acid, biomaterial, nano particle, spin labeling, fluorophore, the containing metal part, radioactive segment, novel functional group, with the covalently or non-covalently interactional group of other molecule, the grating part, but the part of photoisomerization, vitamin H, biotin derivative, the vitamin H analogue, the part of incorporating heavy atom into, but the group of chemical cracking, but the group of photo-cleavage, prolong side chain, the sugar that connects carbon, redox active agent, amino thioic acid sulfoacid, the toxicity part, isotope-labeled part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the intensive group of electronics, the magnetic group, insert group, chromophoric group, energy transfer agent, biologically active agent, detectable label, any combination of small molecules or above material, perhaps any other wanted compound or material.Illustrative limiting examples as composition as described herein, method, technology and strategy, below describe to concentrate on high polymer is made an addition to non-natural amino acid polypeptides, should be appreciated that simultaneously described in addition composition, method, technology and strategy also can be used for (in case of necessity through suitably modification and those skilled in the art can utilize this paper disclosure to carry out) and add other functional group who includes, but is not limited to above listed person.

Multiple high polymer and other molecule can be connected in hGH polypeptide of the present invention and provide new biological characteristics with the biological characteristics of regulating described hGH polypeptide and/or for described hGH molecule.These high polymers can be via natural coded amino acid, be connected in described hGH polypeptide via any function substituting group of non-naturally encoded amino acids or natural or alpha-non-natural amino acid or any substituting group or the functional group that make an addition to natural or alpha-non-natural amino acid.

The invention provides the polymkeric substance of homogeneous substantially: the preparation of albumen joiner." homogeneous substantially " means and observe polymkeric substance as used herein: albumen joiner molecule is greater than half of total protein.Described polymkeric substance: albumen joiner biologically active and the present invention's provided herein " homogeneous substantially " Pegylation hGH polypeptide formulations is enough homogeneous with the preparation of the advantage that shows homogeneous preparation, described advantage for example batch between ease for use in the clinical application in the predictability of pharmacokinetics.

People also can select to prepare polymkeric substance: the mixture of albumen joiner molecule, and advantage provided herein is that people can select single polymkeric substance: the ratio of albumen joiner is to be included in the described mixture.Therefore, in case of necessity, people can prepare various albumen and various quantity attached polymer moieties (promptly two-, three-, four-etc.) mixture and with single polymkeric substance of described joiner and described use method preparation of the present invention: albumen joiner combination, and have single polymkeric substance with predetermined proportion: the mixture of albumen joiner.

Selected polymkeric substance can be water miscible so that the albumen that it connected does not precipitate in the aqueous environments such as physiological environment.Polymkeric substance can be branch or not branched.Use the end product preparation for therapeutic, polymkeric substance is preferably pharmaceutically acceptable.

The ratio of peg molecule and protein molecular will change to some extent, and its concentration in reaction mixture also will change to some extent.Generally speaking, best ratio (with regard to efficient, because have minimal excessive unreacted albumen or polymkeric substance) can be determined by the molecular weight of selected polyoxyethylene glycol and the quantity of available reactive group.When relating to molecular weight, the molecular weight of common described polymkeric substance is high more, and it is just few more then may to be connected in proteic polymer molecule quantity.Similarly, when optimizing these parameters, should consider the branch of described polymkeric substance.Usually, molecular weight high more (or branch is many more), then polymkeric substance: Protein ratios is high more.

As used herein, and when containing PEG:hGH polypeptide joiner, term " significant quantity in the treatment " means the amount that generation provides the Hematocrit of benefit to increase to the patient.Described amount will be in the multiple factor that changes and will depend on the potential cause of disease of the overall physical qualification that comprises the patient and anaemia one by one between body and another individuality.For example, the treatment significant quantity of hGH polypeptide that is used to suffer from the patient of chronic renal failure istime 50 to 150 units/kg on every Wendesdays.The volume production of the hGH polypeptide that is used for the treatment of is given birth to the speed of acceptable Hematocrit increase and described Hematocrit is maintained useful level (usually at least about 30% and usually in 30% to 36% scope).The treatment significant quantity of composition of the present invention is easy to use the material and the program that can openly obtain to determine by the those skilled in the art.

Described water-soluble polymers can be linearity, bifurcated or the branched any structure form of including, but is not limited to.Usually, described water-soluble polymers is poly-(aklylene glycol), such as poly-(ethylene glycol) (PEG), but also can use other water-soluble polymers.For example, use PEG to describe some embodiment of the present invention.

PEG is well-known water-soluble polymers, and it can be buied and maybe can pass through according to well-known method ring-opening polymerization ethylene glycol (Sandier and Karo, Polymer Synthesis in the affiliated field, Academic Press, New York, the 3rd volume, 138-161 page or leaf).Be extensive use of that any peg molecule contained in term " PEG " and no matter the modification of size or PEG end, and can be expressed from the next to being connected in the hGH polypeptide:

XO-(CH₂CH₂O)_n-CH₂CH₂-Y，

Wherein n be 2 to 10,000 and X be H or include, but is not limited to C_1-4Alkyl end modified.

In some cases, be used for PEG of the present invention and end at an end with hydroxyl or methoxyl group, promptly X is H or CH₃(" methoxyl group PEG ").Perhaps, PEG can stop by reactive group, forms the double functional copolymer thus.The type reaction base can comprise that those reactive groups that are generally used for and are found in 20 kinds of functional group's reactions in the common amino acid (include but not limited to maleimide groups, activated carbonate (including but not limited to p-nitrophenyl ester), Acibenzolar (includes but not limited to N-maloyl imines, p-nitrophenyl ester) and aldehyde) and 20 kinds of common amino acids be inertia but (include but not limited to azido-with the functional group who is present in the complementary functional group's specific reaction in the non-naturally encoded amino acid, alkynyl).It should be noted that and be showed in by Y that another of PEG of following formula is terminal will directly or via natural generation or non-naturally encoded amino acids to be connected in the hGH polypeptide indirectly.For example, Y can be acid amides, carbamate or the urea bonding with the amine groups (including but not limited to the ε amine or the N-terminal of Methionin) of polypeptide.Perhaps, Y can be the maleimide bonding with thiol group (including but not limited to the thiol group of halfcystine).Perhaps, Y can be with usually can not be via the bonding of the approaching residue of 20 kinds of common amino acids.For example, the azido-on the PEG can react with the alkynyl on the hGH polypeptide to form Huisgen[3+2] the cycloaddition product.Perhaps, the alkynyl on the PEG can react to form similar product with the azido-that is present in non-naturally encoded amino acids.In certain embodiments, where applicable, strong nucleophilic reagent (including, but is not limited to hydrazine, hydrazides, azanol, Urea,amino-) can form hydrazone, oxime or semicarbazone with the aldehydes or ketones radical reaction that is present in the non-naturally encoded amino acids, and it can further reduce by handling with suitable reductive agent in some cases.Perhaps, described strong nucleophilic reagent can be incorporated the hGH polypeptide into and is used for preferably and ketone that is present in water-soluble polymers or aldehyde radical reaction via non-naturally encoded amino acids.

In case of necessity, can use any molecular mass of PEG in the practice, it includes, but is not limited to about 100 dalton (Da) to 100,000 dalton or higher (including, but is not limited to be sometimes 0.1-50kDa or 10-40kDa) in case of necessity.Also can use and include, but is not limited to each chain and have the side chain PEG of scope as the PEG molecule of the molecular weight of 1-100kDa (including, but is not limited to 1-50kDa or 5-20kDa).The PEG molecule is described in (including, but is not limited to) the Shearwater Polymers widely, and in Inc. catalogue, the Nektar Therapeutics catalogue, it is incorporated herein by reference.

Usually, at least one end of PEG molecule can be used for reacting with non-naturally encoded amino acids.For example, be used for can be used for PEG is connected in non-naturally encoded amino acids as described herein with the PEG derivative with alkynes and nitrine part of amino acid side chain reaction.If non-naturally encoded amino acids comprises azido-, then PEG contains alkynyl moiety usually realize to form [3+2] the cycloaddition product that contains phosphino-or activated PEG material (being ester, carbonic ether) to realize forming amide linkage.Perhaps, if described non-naturally encoded amino acids comprises alkynes, then PEG contains the nitrine part usually to realize forming [3+2] Huisgen cycloaddition product.If non-naturally encoded amino acids comprises carbonyl, then PEG comprises effective nucleophilic group (including, but is not limited to hydrazides, hydrazine, azanol or Urea,amino-functional group) usually to realize forming respectively corresponding hydrazone, oxime and semicarbazone bonding.In other substituting situation, can use with the side of above-mentioned reactive group in the opposite direction, promptly the part of the nitrine in the non-naturally encoded amino acids can with the PEG derivatives reaction that contains alkynes.

In certain embodiments, the hGH polypeptide variants with PEG derivative contain can with the chemical functional group of chemical functional group's reaction on the side chain that is present in non-naturally encoded amino acids.

In certain embodiments, the invention provides the polymer derivant that contains nitrine and acetylene, it comprises and has about 800Da to about 100, the water-soluble polymers skeleton of the molecular-weight average of 000Da.The polymer backbone of water-soluble polymers can be poly-(ethylene glycol).Yet, should be appreciated that, the multiple water-soluble polymers that includes, but is not limited to polyoxyethylene glycol and other related polymer (comprise poly-(dextran) and poly-(propylene glycol)) also is applicable to puts into practice the present invention, and uses term PEG or poly-(ethylene glycol) to be intended to contain and comprise all these molecules.Term PEG includes, but is not limited to be its any type of poly-(ethylene glycol), the PEG that it comprises difunctionality PEG, multi-arm PEG, the PEG that derives, bifurcated PEG, branch PEG, the PEG that dangles (PEG or the related polymer that promptly have one or more functional groups that overhang polymer backbone) or wherein has the degradable bonding.

PEG normally transparent, colourless, tasteless, water soluble, to thermally-stabilised, to number of chemical agent inertia, not hydrolysis or deterioration and nontoxic usually.Think that poly-(ethylene glycol) for biocompatibility, that is to say that PEG can and not cause infringement with living tissue or organism coexistence.More particularly, PEG is a non-immunogenic substantially, that is to say that PEG does not have the immunoreactive tendency of generation in vivo.When be connected in such as biologically active agent have the branch period of the day from 11 p.m. to 1 a.m that some want function in vivo, thereby PEG tends to cover described medicament and can reduce or eliminate the existence that any immune response organism can tolerate described medicament.The PEG joiner tends to not produce the immune response of essence or causes and condenses or other undesirable effect.Has formula-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂The PEG of-(wherein n is about 3 to about 4000, is generally about 20 to about 2000) is applicable to the present invention.In some embodiments of the invention, have about 800Da to about 100, the PEG of the molecular weight of 000Da especially is suitable for and makes polymer backbone.

Polymer backbone can be linear or branched.The branched polymers skeleton is normally known in affiliated field.Branched polymers has branch core, center and a plurality of linear polymer chain that is connected in described center branch core usually.PEG uses with the branch form usually, and it can be by adding to oxyethane such as preparation on the various polyvalent alcohols of glycerine, glycerine oligomer, tetramethylolmethane and Sorbitol Powder.Center branch part also can be derived from several amino acid such as Methionin.Branch poly-(ethylene glycol) can be as R (PEG-OH)_mThe general type of (wherein R is derived from the core, and such as glycerine, glycerine oligomer or tetramethylolmethane, and m represents the quantity of arm) is represented.Also can be used as polymer backbone such as the multi-arm PEG molecule that is described in the following document: United States Patent (USP) the 5th, 932,462,5,643,575,5,229,490,4,289, No. 872, U.S. patent application case 2003/0143596, WO 96/21469 and WO 93/21259, its mode of quoting in full separately is incorporated herein.

Branch PEG also can be for by PEG (YCHZ₂)_nThe form of the bifurcated PEG of (wherein Y is for connecting base and Z for be connected in the activation end group of CH by the atomchain of determining length) expression.

Another branch form PEG that dangles has reactive group such as carboxyl along the PEG skeleton rather than at the end of PEG chain.

Except that these PEG forms, also can prepare polymkeric substance a little less than in skeleton, having or the degradable bonding.For example, can prepare the PEG that in polymer backbone, has the ester bond that stands hydrolysis.Shown in hereinafter, it is the fragment of lower molecular weight that this hydrolytic action causes polymer cracking:

It will be understood by one of ordinary skill in the art that term poly-(ethylene glycol) or PEG represent or comprise under all known form of ownership in fields, it includes, but is not limited to person disclosed herein.

Multiple other polymkeric substance also is applicable to the present invention.In certain embodiments, have 2 water-soluble polymers skeletons and be particularly useful for the present invention to about 300 ends.Suitably the example of polymkeric substance includes, but is not limited to other poly-(aklylene glycol), such as poly-(propylene glycol) (" PPG "), its multipolymer (including, but is not limited to the multipolymer of ethylene glycol and propylene glycol), its trimer, its mixture or the like.Although the molecular weight of each chain of polymer backbone can change, its usually at about 800Da to about 100,000Da, be generally about 6,000Da is to about 80, in the scope of 000Da.

Those of ordinary skill in the field will recognize, the previous list of water miscible skeleton is detailed absolutely not and only be illustrative substantially, and expect that all polymeric materials with above-mentioned quality all are applicable to the present invention.

In some embodiments of the invention, polymer derivant is " polyfunctional ", and it means polymer backbone and has functionalized or at least two ends of activatory through the functional group, and may be up to about 300 ends.Multifunctional polymer derivant includes, but is not limited to have the linear polymer of two ends, wherein each end be binding on can be identical or different the functional group.

In one embodiment, polymer derivant has following structure:

X-A-POLY-B-N＝N＝N

Wherein:

N=N=N is the nitrine part;

B is the connection portion, and it can exist or not exist:

POLY is water-soluble nonantigenic polymkeric substance;

A is the connection portion, and it can exist or not exist and can be identical or different with B; And

X is second functional group.

The example of the connection portion of A and B includes, but is not limited to contain up to 18 and the multiple functionalized alkyl of the carbon atom between 1-10 more preferably.Heteroatoms such as nitrogen, oxygen or sulphur can be included in the alkyl chain.Alkyl chain also can be at the heteroatoms upper branch.Other example of the connection portion of A and B includes, but is not limited to contain up to 10 and the multiple functionalized aryl of 5-6 carbon atom more preferably.Aryl can replace through one or more carbon atoms, nitrogen, oxygen or sulphur atom.Suitable other example that connects base comprises and is described in United States Patent (USP) the 5th, 932,462,5,643, and No. 575 and in the U.S. Patent Application Publication case 2003/0143596 (it is incorporated herein by reference separately) those are connected basic.Those of ordinary skill in the field will recognize, the above-mentioned tabulation of connection portion is detailed absolutely not and only be illustrative, and expect that all connection portions with above-mentioned quality all are applicable to the present invention.

The example that is used as the suitable functional group of X includes, but is not limited to hydroxyl; hydroxyl and protected; alkoxyl group; active ester such as N-maloyl imido grpup ester and 1-benzotriazole base ester; activated carbon acid esters such as carbonic acid N-maloyl imido grpup ester and carbonic acid 1-benzotriazole base ester; acetal; aldehyde; aldehyde hydrate; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; aminooxy; shielded amine; hydrazides; shielded hydrazides; shielded mercaptan; carboxylic acid; shielded carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulphone; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate; the trifluoro esilate; alkene; ketone and trinitride.It will be understood by one of ordinary skill in the art that thereby the reaction with azido-should compatiblely with azido-not take place selected X part.The polymer derivant that contains nitrine can be with the difunctionality base, it means second functional group (being X) and also is the nitrine part, or is the Heterobifunctional base, and it means second functional group is different functional groups.

Term " shielded " means to exist and prevent protecting group or the part that the chemical reactivity functional group reacts under some reaction conditions.Protecting group will be looked the type of shielded chemical reaction base and be changed.For example, if the chemical reaction base is amine or hydrazides, then protecting group can be selected from the group of tertbutyloxycarbonyl (t-Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc).If described chemical reaction base is a mercaptan, then protecting group can be adjacent pyridyl disulfide.If the chemical reaction base is such as the carboxylic acid of butyric acid or propionic acid or hydroxyl, then protecting group can be phenmethyl or such as the alkyl of methyl, ethyl or the tertiary butyl.Known other protecting group also can be used for the present invention in the affiliated field.

Terminal functional group's particular instance includes, but is not limited to carbonic acid N-succimide ester (for example referring to United States Patent (USP) the 5th in the document, 281,698,5,468, No. 478), amine (for example referring to people such as Buckmann, Makromol.Chem.182:1379 (1981); People such as Zaplipsky, Eur.Polym.J.19:1177 (1983)), hydrazides (for example referring to people Makromol.Chem.179:301 (1978) such as Andresz), propionic acid succimide base ester and butyric acid succimide base ester (for example referring to people such as Olson in Poly (ethylene glycol) Chemistry ﹠amp; Biological Applications, 170-181 page or leaf, Harris ﹠amp; Zaplipsky Eds., ACS, Washington, D.C., 1997; Also referring to United States Patent (USP) the 5th, 672, No. 662), Succinic Acid succimide base ester (for example referring to people Macrolol.Chem.180:1381 (1979) such as people Cancer Biochem.Biophys.7:175 (1984) such as Abuchowski and Joppich), succimide base ester be (for example referring to United States Patent (USP) the 4th, 670, No. 417), the benzotriazole carbonic ether is (for example referring to United States Patent (USP) the 5th, 650, No. 234), glycidyl ether (for example referring to people such as Pitha, Eur.J Biochem.94:11 (1979); People such as Elling, Biotech.Appl.Biochem.13:354 (1991)), the oxygen carbonylic imidazole (for example referring to people such as Beauchamp, Anal.Biochem.131:25 (1983); People such as Tondelli, J.Controlled Release 1:251 (1985)), p-nitrophenyl carbonate ester (for example referring to people such as Veronese, Appl.Biochem.Biotech., 11:141 (1985); With people such as Sartore, Appl.Biochem.Biotech., 27:45 (1991)), aldehyde (for example referring to people such as Harris, J.Polym.Sci.Chem.Ed.22:341 (1984); United States Patent (USP) the 5th, 824, No. 784; United States Patent (USP) the 5th, 252, No. 714), maleimide is (for example referring to people such as Goodson, Bio/Technology 8:343 (1990), people such as Romani, Chemistry of Peptidesand Proteins 2:29 (1984)) and Kogan, Synthetic Comm.22:2417 (1992)), adjacent pyridyl disulfide is (for example referring to people such as Woghiren, Bioconj.Chem.4:314 (1993)), vinylcarbinol is (for example referring to people such as Sawhney, Macromolecules, 26:581 (1993)), vinyl sulphone (for example referring to United States Patent (USP) the 5th, 900, No. 461).All above reference and patent all are incorporated herein by reference.

In certain embodiments of the present invention, polymer derivant of the present invention comprises the polymer backbone with following structure:

X-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-N＝N＝N

Wherein:

X is above-mentioned functional group; And

N is about 20 to about 4000.

In another embodiment, polymer derivant of the present invention comprises the polymer backbone with following structure:

X-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-O-(CH₂)_m-W-N＝N＝N

Wherein:

W is aliphatic series or the aromatics connection portion that comprises the carbon atom between 1-10;

N is about 20 to about 4000; And

X is above-mentioned functional group.M is between 1 and 10.

The PEG derivative that contains nitrine of the present invention can under several different methods preparation known and/or disclosed herein in the field.In a kind of method shown below, make to have about 800Da the water-soluble polymers skeleton of the molecular-weight average of 000Da (described polymer backbone have be binding on the first terminal and be binding on second end of suitable leaving group of first functional group) and nitrine negatively charged ion (its can with any pairing in the multiple proper equilibrium ion that comprises sodium, potassium, uncle's fourth ammonium or the like) reaction to about 100.Leaving group experiences nucleophilic displacement and is partly replaced by nitrine, thereby the PEG that will contain nitrine is provided polymkeric substance.

As shown, be used for suitable polymer backbone of the present invention and have formula X-PEG-L, wherein PEG is that poly-(ethylene glycol) and X are to be not suitable leaving group with the functional group and the L of azido-reaction.Suitably functional group's example includes, but is not limited to hydroxyl, hydroxyl and protected, acetal, thiazolinyl, amine, aminooxy, shielded amine, shielded hydrazides, shielded mercaptan, carboxylic acid, shielded carboxylic acid, maleimide, two thiopyridines and vinyl pyridine and ketone.The example of suitable leaving group includes, but is not limited to muriate, bromide, iodide, methanesulfonate, trifluoro ethyl sulfonic acid root and tosylate.

Be used for preparing the other method that contains the azide polymer derivative of the present invention, make linking agent contact have about 800Da to about 100 with nitrine functional group, the water-soluble polymers skeleton of the molecular-weight average of 000Da, contain the azide polymer derivative products to form wherein azido-and polymer backbone by what be connected that base separates, wherein said linking agent have with the PEG polymkeric substance on the chemical functional group of chemical functional group's selective reaction.

Exemplary reaction process is showed as follows:

Wherein:

PEG is that poly-(ethylene glycol) and X are the capping groups such as alkoxyl group or above-mentioned functional group; And

M is not with nitrine functional group reaction but the functional group of and selective reaction effective with the N functional group.

Suitably functional group's example includes, but is not limited to: if N is an amine, then M is carboxylic acid, carbonic ether or active ester; If N is hydrazides or aminooxy part, then M is a ketone; If N is a nucleophilic group, then M is a leaving group.

Can include, but is not limited to precipitated product and carry out the purifying that the stratographic currently known methods is finished raw product in case of necessity subsequently.

More particular instance is showed in the situation of following PEG diamines, and one of wherein said amine is by partly protecting such as the protecting group of the tertiary butyl-Boc and gained list protection PEG diamines and the connection portion reaction with nitrine functional group:

BocHN-PEG-NH₂+HO₂C-(CH₂)₃-N＝N＝N。

In this case, can use such as the multiple activator of thionyl chloride or carbon diimide reagent and N-maloyl imines or N-hydroxybenzotriazole amine groups and hydroxy-acid group coupling with at monoamine PEG derivative and have between the connexon part of nitrine and produce amido linkage.After successfully forming amido linkage, can directly use gained to contain azido derivant modified biological bioactive molecule or can be further that it is refining so that other useful functional group to be installed through the protection of the N-tertiary butyl-Boc.For example, can be by come hydrolyzing N-t-Boc group with strong acid treatment to produce omega-amino--PEG-trinitride.Gained amine can be used as synthetic handle with install other such as the useful functional group of maleimide groups, activatory disulphide, Acibenzolar or the like to produce valuable Heterobifunctional reagent.

When hope in that each of polymkeric substance is terminal when connecting differing molecular, the Heterobifunctional derivative is particularly useful.For example, ω-N-amino-N-azido-PEG allows to connect molecule with activation electrophilic group (such as aldehyde, ketone, Acibenzolar, activated carbonate or the like) and the molecule that has acetylene group in another terminal connection of PEG at the end of PEG.

In another embodiment of the present invention, polymer derivant has following structure:

X-A-POLY-B-C≡C-R

Wherein:

The aryl that R can be H or alkyl, alkene, alkoxyl group or aryl or is substituted;

B is the connection portion, and it can exist or not exist;

POLY is water-soluble nonantigenic polymkeric substance;

A is the connection portion, and it can exist or not exist and it can be identical or different with B; And

X is second functional group.

The example of the connection portion of A and B includes, but is not limited to contain up to 18 and the multiple functionalized alkyl of the carbon atom between 1-10 more preferably.Heteroatoms such as nitrogen, oxygen or sulphur can be included in the alkyl chain.Alkyl chain also can be at the heteroatoms upper branch.Other example of the connection portion of A and B includes, but is not limited to contain up to 10 and the multiple functionalized aryl of 5-6 carbon atom more preferably.Described aryl can replace through one or more carbon atoms, nitrogen, oxygen or sulphur atom.Suitable other example that connects base comprises and is described in United States Patent (USP) the 5th, 932,462,5,643, and No. 575 and in the U.S. Patent Application Publication case 2003/0143596 (it is incorporated herein by reference separately) those are connected basic.Those of ordinary skill in the field will recognize, the above-mentioned tabulation of connection portion is detailed absolutely not and only be illustrative, and expect that multiple connection portion with above-mentioned quality is applicable to the present invention.

The example that is used as the suitable functional group of X comprises hydroxyl; hydroxyl and protected; alkoxyl group; active ester such as N-maloyl imido grpup ester and 1-benzotriazole base ester; activated carbon acid esters such as carbonic acid N-maloyl imido grpup ester and carbonic acid 1-benzotriazole base ester; acetal; aldehyde; aldehyde hydrate; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; aminooxy; shielded amine; hydrazides; shielded hydrazides; shielded mercaptan; carboxylic acid; shielded carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulphone; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate; the trifluoro esilate; alkene; ketone and acetylene.Should be appreciated that, thereby the reaction with described acetylene group should compatiblely with acetylene group not take place in selected X part.The polymer derivant that contains acetylene can be with the difunctionality base, it means second functional group (being X) and also is acetylene moiety, or is the Heterobifunctional base, and it means second functional group is different functional groups.

In another embodiment of the present invention, polymer derivant comprises the polymer backbone with following structure:

X-CH₂CH₂O-(CH₂CH₂O)_n-CH₂CH₂-O-(CH₂)_m-C ≡ CH is wherein:

X is above-mentioned functional group;

N is about 20 to about 4000; And

M is between 1 and 10.

The particular instance of each Heterobifunctional base PEG polymkeric substance is showed in hereinafter.

The PEG derivative that contains acetylene of the present invention can use those skilled in the art's method preparation known and/or disclosed herein.In one approach, make to have about 800Da to about 100, the water-soluble polymers skeleton of the molecular-weight average of 000Da (described polymer backbone have be binding on the first terminal and be binding on second end of suitable nucleophilic group of first functional group) with have the acetylene functional group and be suitable for PEG on the compound reaction of leaving group of nucleophilic group reaction.When having nucleophilic PEG polymkeric substance partly and having the molecular combinations of leaving group, described leaving group experiences nucleophilic displacement and is partly replaced by described nucleophilic, will contain acetylene polymer thereby provide.

As demonstrated, the preferred polymers skeleton that is used to react has formula X-PEG-Nu, and wherein PEG is poly-(ethylene glycol), Nu be nucleophilic part and X for not with the functional group of Nu, L or acetylene functional group reaction.

The example of Nu includes, but is not limited to mainly via machine-processed amine, alkoxyl group, aryloxy, sulfydryl, imido grpup, carboxylicesters, hydrazides, the aminooxy that reacts of SN2 type.Other example of Nu group comprises those functional groups that mainly react via nucleophilic addition.The L examples of groups comprises the group of muriate, bromide, iodide, methanesulfonate, trifluoro ethyl sulfonic acid root and tosylate and other expection experience nucleophilic displacement and the electrophilic group of ketone, aldehyde, thioesters, alkene, alpha-beta unsaturated carbonyl, carbonic ether and other expection experience nucleophilic reagent addition.

In another embodiment of the present invention, A is the aryl rings that is substituted with the aliphatic connexon of 1-10 carbon atom or 6-14 carbon atom.X is to be not suitable leaving group with the functional group and the L of azido-reaction.In the preparation other method that contains the acetylene polymer derivative of the present invention; make to have about 800Da, the molecular-weight average of 000Da, have shielded functional group or end-capping reagent contacts the acetylene negatively charged ion with the PEG polymkeric substance that has suitable leaving group at another end at an end to about 100.

Exemplary reaction process is showed as follows:

Wherein:

PEG is that poly-(ethylene glycol) and X are the capping groups such as alkoxyl group or above-mentioned functional group; And

R ' is H, alkyl, alkoxyl group, aryl or aryloxy or the alkyl that is substituted, alkoxyl group, aryl or aryloxy.

In above example, leaving group L should have enough reactivities with experience SN2 type replacement(metathesis)reaction when contacting the acetylene negatively charged ion of enough concentration.Finishing by the required reaction conditions of acetylene negatively charged ion SN2 displacement leaving group is well-known in affiliated field.

Usually can be by including, but is not limited to precipitated product and carrying out under the stratographic purifying that known method in the field is finished raw product where necessary subsequently.

Water-soluble polymers can be connected in hGH polypeptide of the present invention.Described water-soluble polymers can connect via any functional group or the substituting group of incorporating the non-naturally encoded or natural coded amino acid of non-naturally encoded amino acids in the hGH polypeptide into or any functional group or the substituting group that add non-naturally encoded or natural coded amino acid.Perhaps, described water-soluble polymers is connected in the hGH polypeptide of incorporating non-naturally encoded amino acids into via natural generation amino acid (including, but is not limited to the amine groups of halfcystine, Methionin or N-terminal residue).In some cases, hGH polypeptide of the present invention comprises 1,2,3,4,5,6,7,8,9,10 alpha-non-natural amino acid, and one of them or an above non-naturally encoded amino acids are connected in water-soluble polymers (including, but is not limited to PEG and/or oligosaccharides).In some cases, hGH polypeptide of the present invention further comprises 1,2,3,4,5,6,7,8,9,10 or more multi-link in the natural coded amino acid of water-soluble polymers.In some cases, hGH polypeptide of the present invention comprises one or more non-naturally encoded amino acids that are connected in water-soluble polymers and one or more are connected in the natural generation amino acid of water-soluble polymers.In certain embodiments, be used for water-soluble polymers of the present invention strengthens described hGH polypeptide with respect to joint form not serum half-life.

The quantity (being Pegylation or glycosylated degree) that can regulate the water-soluble polymers that is connected in hGH polypeptide of the present invention is with pharmacology, pharmacokinetics or the pharmacodynamic profile of (include, but is not limited to increase or reduce) such as transformation period in the body that change is provided.In certain embodiments, the transformation period of hGH increases at least aboutpercent 10,20,30,40,50,60,70,80,90,2 times, 5 times, 10 times, 50 times or at least about 100 times than unmodified polypeptide.

The PEG derivative that contains strong nucleophilic group (being hydrazides, hydrazine, azanol or Urea,amino-)

In one embodiment of the invention, comprising the hGH polypeptide that contains the carbonyl non-naturally encoded amino acids modifies through the PEG derivative that contains the terminal hydrazine, azanol, hydrazides or the Urea,amino-part that are directly connected in the PEG skeleton.

In certain embodiments, the terminal PEG derivative of azanol has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-O-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (being that molecular-weight average is between 5-40kDa).

In certain embodiments, contain hydrazine or hydrazides PEG derivative has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-X-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, contain Urea,amino-PEG derivative and have following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-NH-C(O)-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, comprise the hGH polypeptide that contains carbonylamino acid and modify through containing the PEG derivative that is connected in terminal azanol, hydrazides, hydrazine or the Urea,amino-part of PEG skeleton by means of amide linkage.

In certain embodiments, the terminal PEG derivative of azanol has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)(CH₂)_m-O-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (being that molecular-weight average is between 5-40kDa).

In certain embodiments, contain hydrazine or hydrazides PEG derivative has following structure:

RO-(CH₂CH₂O)_n-O-(CH2)2-NH-C(O)(CH₂)_m-X-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2-10, and n is 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, contain Urea,amino-PEG derivative and have following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)(CH₂)_m-NH-C(O)-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, comprise the hGH polypeptide that contains carbonylamino acid and modify through the branch PEG derivative that contains terminal hydrazine, azanol, hydrazides or Urea,amino-part, each bar chain of wherein said branch PEG has scope to be 10-40kDa and more preferably to be the MW of 5-20kDa.

In another embodiment of the present invention, the PEG derivative of hGH polypeptide through having apparatus derivatorius that comprises non-naturally encoded amino acids modified.For example, in certain embodiments, the terminal PEG derivative of hydrazine or hydrazides has following structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)]₂CH(CH₂)_m-X-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 and X according to circumstances for can exist or non-existent carbonyl (C=O).

In certain embodiments, the PEG derivative that contains the Urea,amino-group has following structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-C(O)-NH-CH₂-CH₂]₂CH-X-(CH₂)_m-NH-C(O)-NH-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or does not exist that m is that 2-10 and n are 100-1 according to circumstances, 000.

In certain embodiments, the PEG derivative that contains the azanol group has following structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-C(O)-NH-CH₂-CH₂]₂CH-X-(CH₂)_m-O-NH₂

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or does not exist that m is that 2-10 and n are 100-1 according to circumstances, 000.

Water-soluble polymers be connected in the degree of hGH polypeptide and site can regulate hGH polypeptide and hGH polypeptide receptor in thesite 1 combine.In certain embodiments, arrange bonding in case the hGH polypeptide with about 400nM or lower K_d, with 150nM or lower K_dAnd in some cases with 100nM or lower K_d(as by such as hGH being described in people such as Spencer, J.Biol.Chem., the balance among the 263:7862-7867 (1988) is measured in conjunction with calibrating) in thesite 1 in conjunction with the hGH polypeptide receptor.

Be used for activated polymer and engage the method for peptide and chemical descriptor in document and known in affiliated field.The common method that is used for activated polymer includes, but is not limited to activation functional groups such as cyanogen bromide, periodate, glutaraldehyde, di-epoxide, Epicholorohydrin, divinylsulfone, carbonization imide, sulfuryl halide, three chlorotriazines (referring to R.F.Taylor, (1991), PROTEIN I_{MMOBILISATION}.F_{UNDAMENTAL AND}A_PPLICATIONS, MarcelDekker, N.Y.; S.S.Wong, (1992), C_{HEMISTRY OF}P_ROTEINC_{ONJUGATION AND}C_ROSSLINKING, CRC Press, Boca Raton; People such as G T.Hermanson, (1993), IMMOBILIZED AFFINITYL1GAND TECHNIQUES, Academic Press, N.Y.; Dunn, people Eds.POLYMERICDRUGS AND DRUG DELIVERY SYSTEMS such as R.L., ACS Symposium Series, the 469th volume, AmericanChemical Society, Washington, D.C.1991).

Can obtain several review and monographs to the functionalized of PEG and joint.For example referring to Harris, Macronol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, Critical Reviews in TherapeuticDrug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995).

The method that is used for activated polymer also is found in WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO90/13540, United States Patent (USP) the 5th, 281, among No. 698 and the WO 93/15189, and also visible activated polymer and include, but is not limited to blood coagulation factor VIII (WO 94/15625), oxyphorase (WO 94/09027), oxygen carrier molecule (United States Patent (USP) the 4th, 412, No. 989), the method of the joint between the enzyme of rnase and superoxide dismutase (people such as Veronese, App.Biochem.Biotech.11:141-45 (1985)).The reference and the patent of all references all are incorporated herein by reference.

It is to carry out with any facilitated method that Pegylation (promptly adding any water-soluble polymers) contains such as the hGH polypeptide to the non-naturally encoded amino acids of azido--L-phenylalanine.For example, use the mPEG derivative Pegylation hGH polypeptide of alkynes terminal.In brief, at room temperature accompany by stirring with excessive solid mPEG (5000)-O-CH₂-C ≡ CH adds in the aqueous solution contain the hGH polypeptide of azido--L-Phe.Usually, near the apparatus pK its pH (about usually pH4-10) that carries out down that responds_aThe damping fluid aqueous buffer solution.For example, the example that is used for the suitable damping fluid of Pegylation under pH7.5 includes, but is not limited to HEPES, phosphoric acid salt, borate, TRIS-HCl, EPPS and TES.Monitoring continuously and regulate pH in case of necessity.Usually allow to react between about continuously 1-48 hour.

Make reaction product stand hydrophobic interaction chromatograph subsequently to separate Pegylation hGH polypeptide variants and free mPEG (5000)-O-CH₂Any high molecular weight component of-C ≡ CH and Pegylation hGH polypeptide, thus when not the blocking-up PEG divide the period of the day from 11 p.m. to 1 a.m can form described high molecular weight component at the activated crosslinked hGH polypeptide variants of two ends of molecule.Condition during the hydrophobic interaction chromatograph should make free mPEG (5000)-O-CH₂-C ≡ CH flows through tubing string, any cross-linked polyethylene glycol hGH polypeptide variants mixture elution after want form simultaneously, and it contains a hGH polypeptide variants molecule that is engaged in one or more PEG groups.Appropriate condition is looked the relative size change of cross-linked composite to want joiner, and is easy to be determined by the those skilled in the art.Contain the elutant of wanting joiner to some extent and pass through ultrafiltration and concentration also by saturating elimination salt.

In case of necessity, described Pegylation hGH polypeptide available from hydrophobic chromatography can further pass through known one or more program purifying of those skilled in the art, and described program comprises (but being not limited to) affinity chromatography, negatively charged ion or cation-exchange chromatography (use includes, but is not limited to DEAE SEPHAROSE), silica gel chromatography, reversed-phase HPLC, gel-filtration (use includes, but is not limited to SEPHADEX G-75), hydrophobic interaction chromatograph, the size exclusion chromatogram, immobilized metal ion afinity chromatography, ultrafiltration/saturating filter, ethanol sedimentation, ammonium sulfate precipitation, chromatofocusing, displcement chromatography, electrophoretic procedures (including, but is not limited to the isoelectrofocusing of preparation type), difference solubleness (including, but is not limited to ammonium sulfate precipitation) or extraction.Apparent molecular weight can by with the sphaeroprotein standard relatively and by the GPC estimation (PROTEIN PURIFICATIONMETHODS, A PRACTICAL APPROACH (Harris and Angal compile) IRL Press 1989,293-306).The purity of hGH-PEG joiner can be assessed by proteolysis type degraded (including, but is not limited to the trypsinase cracking) mass spectroscopy subsequently.People such as Pepinsky B., J.Pharmcol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001).

Can further unrestrictedly derive or replace the amino acid whose water-soluble polymers that is connected in hGH polypeptide of the present invention.

The PEG derivative that contains nitrine

In another embodiment of the present invention, repair hGH polypeptide decorations with the PEG derivative that contains with the nitrine part that is present in the alkynyl moiety reaction on the non-naturally encoded amino acids side chain.Usually, the PEG derivative have scope be 1-100kDa and in certain embodiments scope be the molecular-weight average of 10-40kDa.

In certain embodiments, the terminal PEG derivative of nitrine has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-N₃

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (being that molecular-weight average is between 5-40kDa).

In another embodiment, the terminal PEG derivative of nitrine has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-NH-C(O)-(CH₂)_p-N₃

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2-10, and p is that 2-10 and n are 100-1,000 (being that molecular-weight average is between 5-40kDa).

In another embodiment of the present invention, comprise the hGH polypeptide that contains alkynyl amino acid and modify through the branch PEG derivative that contains terminal nitrine part, each bar chain of wherein said branch PEG has scope to be 10-40kDa and more preferably to be the MW of 5-20kDa.For example, in certain embodiments, the terminal PEG derivative of nitrine has following structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)]₂CH(CH₂)_m-X-(CH₂)pN₃

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2-10, and p is that 2-10 and n are 100-1,000, and X is O, N, S or carbonyl (C=O) according to circumstances, X can exist or not exist under each situation.

Contain alkynes PEG derivative

In another embodiment of the present invention, modify the hGH polypeptide with the PEG derivative that contains with the alkynyl moiety that is present in the nitrine partial reaction on the non-naturally encoded amino acids side chain.

In certain embodiments, the terminal PEG derivative of alkynes has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-C≡CH

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (being that molecular-weight average is between 5-40kDa).

In another embodiment of the present invention, comprise the hGH polypeptide that contains the alkynes non-naturally encoded amino acids and be connected in the terminal nitrine part of PEG skeleton or the PEG derivative modification of terminal alkynyl moiety through containing by means of amide linkage.

In certain embodiments, the terminal PEG derivative of alkynes has following structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-NH-C(O)-(CH₂)_p-C≡CH

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2-10, and p is that 2-10 and n are 100-1,000.

In another embodiment of the present invention, comprise and contain the amino acid whose hGH polypeptide of nitrine and modify through the branch PEG derivative that contains terminal alkynyl moiety, each bar chain of wherein said branch PEG has scope to be 10-40kDa and more preferably to be the MW of 5-20kDa.For example, in certain embodiments, the terminal PEG derivative of alkynes has following structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-NH-C(O)]₂CH(CH₂)_m-X-(CH₂)_pC≡CH

Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is 2-10, and p is that 2-10 and n are 100-1,000, and X is O, N, S or carbonyl (C=O) according to circumstances or does not exist.

Contain phosphine PEG derivative

In another embodiment of the present invention, further comprise and the aryl phosphine that is present in the nitrine partial reaction on non-naturally encoded amino acids side chain PEG derivative modification hGH polypeptide partly with containing activation functional group (including, but is not limited to ester, carbonic ether).Usually, the PEG derivative have scope be 1-100kDa and in certain embodiments scope be the molecular-weight average of 10-40kDa.

In certain embodiments, described PEG derivative has following structure:

Wherein n is 1-10, and X can be O, N, S or do not exist, and Ph is that phenyl and W are water-soluble polymers.

In certain embodiments, described PEG derivative has following structure:

Wherein X can be O, N, S or does not exist, and Ph is a phenyl, and W is the aryl that water-soluble polymers and R can be H, alkyl, aryl, the alkyl that is substituted and be substituted.Exemplary R group includes, but is not limited to-CH₂,-C (CH₃)₃,-OR ' ,-NR ' R " ,-SR ' ,-halogen ,-C (O) R ' ,-CONR ' R " ,-S (O)₂R ' ,-S (O)₂NR ' R " ,-CN and-NO₂R ', R ", R  and R " " the assorted alkyl that means hydrogen independently of one another, is substituted or is unsubstituted, the aryl that is substituted or is unsubstituted (include but not limited to replace aryl), alkyl, alkoxyl group or the thioalkoxy group or the arylalkyl that are substituted or are unsubstituted through 1-3 halogen.When compound of the present invention comprised an above R group, for example, each R group all was independent selection, when having R ', R ", R  and R " " when one of group is above, each R ', R ", R  and R " " the group situation is the same.As R ' and R " when being connected in identical nitrogen-atoms, it can make up to form 5,6 or 7 yuan of rings with nitrogen-atoms.For example ,-and NR ' R " mean and include, but is not limited to 1-pyrrolidyl and 4-morpholinyl.According to substituent above the discussion, be understood by those skilled in the art that term " alkyl " means and comprise following group: it comprises the carbon atom that is attached to the group outside the dehydrogenation base, (includes but not limited to-CF such as haloalkyl₃With-CH₂CF₃) and acyl group (include but not limited to-C (O) CH₃,-C (O) CF₃,-C (O) CH₂OCH₃Or the like).

Other PEG derivative and general Pegylation technology

Other can be connected in the exemplary PEG molecule of hGH polypeptide and Pegylation method and comprise and be described in following document person: for example the U.S. Patent Publication case the 2004/0001838th, 2002/0052009,2003/0162949,2004/0013637,2003/0228274,2003/0220447,2003/0158333,2003/0143596,2003/0114647,2003/0105275,2003/0105224,2003/0023023,2002/0156047,2002/0099133,2002/0086939,2002/0082345,2002/0072573,2002/0052430,2002/0040076,2002/0037949,2002/0002250,2001/0056171,2001/0044526,2001/0027217, No. 2001/0021763, United States Patent (USP) the 6th, 646,110,5,824,778,5,476,653,5,219,564,5,629,384,5,736,625,4,902,502,5,281,698,5,122,614,5,473,034,5,516,673,5,382,657,6,552,167,6,610,281,6,515,100,6,461,603,6,436,386,6,214,966,5,990,237,5,900,461,5,739,208,5,672,662,5,446,090,5,808,096,5,612,460,5,324,844,5,252,714,6,420,339,6,201,072,6,451,346,6,306,821,5,559,213,5,612,460,5,747,646,5,834,594,5,849,860,5,980,948,6,004,573,6,129, No. 912, WO 97/32607, EP 229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO95/13090, WO 95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510 356, EP 400 472, EP 183 503 and EP 154 316, it is incorporated herein by reference.Any PEG molecule as herein described can use in any form, and described form includes, but is not limited to strand, branched chain, multi-arm chain, simple function, difunctionality, multifunctional or its any combination.

Enhancing is to sero-abluminous avidity

Also various molecules can be blended in hGH polypeptide of the present invention to regulate the hGH transformation period of polypeptide in serum.In certain embodiments, molecule is connected or be blended in hGH polypeptide of the present invention to strengthen to endogenous sero-abluminous avidity in the animal.

For example, in some cases, the reorganization syzygy of preparation hGH polypeptide and albumin bound sequence.Exemplary albumin bound sequence includes, but is not limited to the albumin bound territory (people such as Makrides from streptococcus protein G, people such as J.Pharmacol.Exp.Ther.277:534-542 (1996) and sjolander, J, Immunol.Methods201:115-123 (1997)) or such as being described in people such as Dennis, the albumin binding peptide among the J.Biol.Chem.277:35035-35043 (2002).

In other embodiments, hGH polypeptide of the present invention is through the lipid acid acidylate.In some cases, lipid acid promotes to combine with sero-abluminous.For example referring to people such as Kurtzhals, Biochem.J.312:725-731 (1995).

In other embodiments, direct and serum albumin (the including, but is not limited to the human serum albumin) fusion of hGH polypeptide of the present invention.It will be understood by one of ordinary skill in the art that multiple other molecule also can be connected in hGH the combining with adjusting and serum albumin or other serum component among the present invention.

The glycosylation of X.hGH polypeptide

The present invention includes the hGH polypeptide of incorporating one or more non-naturally encoded amino acids into saccharide residue.Saccharide residue can be natural (including, but is not limited to the N-acetyl glucosamine) or non-natural (including, but is not limited to 3-fluorine semi-lactosi).Described sugar can be connected in non-naturally encoded amino acids by glucosidic linkage (including, but is not limited to N-ethanoyl semi-lactosi-L-Serine) or the non-natural bonding (including, but is not limited to the C-of oxime or correspondence or the glycoside that S-connects) that N-or O-connect.

Can be in vivo or external sugar (including, but is not limited to glycosyl) part is added on the hGH polypeptide.In some embodiments of the invention, comprise the hGH polypeptide that contains the carbonyl non-naturally encoded amino acids through sugar-modified to produce the corresponding corresponding glycosylated polypeptides that connects via the oxime bonding with the aminooxy deutero-.In case be connected with non-naturally encoded amino acids, described sugar can further be made with extra care the oligosaccharides that is incorporated into the hGH polypeptide with generation by handling with glycosyltransferase and other enzyme.For example referring to people such as H.Liu, J.Am.Chem.Soc.US:1702-1703 (2003).

In some embodiments of the invention, comprising the hGH polypeptide that contains the carbonyl non-naturally encoded amino acids directly modifies through having the glycan that the regulation structure is prepared as the aminooxy derivative.It will be understood by one of ordinary skill in the art that comprise trinitride, alkynes, hydrazides, hydrazine and Urea,amino-other functional group can be used for described sugar is connected in non-naturally encoded amino acids.

In some embodiments of the invention, the hGH polypeptide that comprises the non-naturally encoded amino acids that contains nitrine or alkynyl then can be by including, but is not limited to respectively and the Huisgen[3+2 that includes, but is not limited to alkynyl or azido derivant] cycloaddition reaction modified.This method allows with high selective modification albumen.

XI.GH supergene family member's dimer and polymer

The present invention also provides GH supergene family member combination (including, but is not limited to hGH) homodimer, heterodimer, homopolymer or heteropolymer (being tripolymer, the tetramer etc.), wherein the GH supergene family member polypeptide such as the hGH that contains one or more non-naturally encoded amino acids is incorporated into another supergene family member or its varient or any other polypeptide for non-GH supergene family member or its varient, and it can directly or via connexon be incorporated into polypeptide backbone.Because molecular weight than its increase of monomer, GH supergene family member's dimer or polymer joiner such as hGH can represent new or desired character, include, but is not limited to different pharmacology, pharmacokinetics, pharmacodynamics, the therapeutical agent transformation period through regulating or the plasma half-life through regulating with respect to monomer GH supergene family member.In certain embodiments, the GH supergene family member dimer such as hGH of the present invention is regulated the dimerisation of GH supergene family member acceptor.In other embodiments, GH supergene family member's dimer of the present invention or polymer serve as GH supergene family member receptor antagonist, agonist or conditioning agent.

In certain embodiments, one or more that are present in the dimer that contains hGH or the hGH molecule in the polymer comprise and are connected in the non-naturally encoded amino acids that is present in the water-soluble polymers in the II calmodulin binding domain CaM of site.Therefore, dimer or polymeric each hGH molecule can near with via I interface, site in conjunction with the hGH polypeptide receptor but be not useable for via II interface, site in conjunction with the 2nd hGH polypeptide receptor.Therefore, the site I binding site of each of engageable two the different hGH polypeptide receptors of hGH polypeptide dimer or polymer, but because the hGH molecule has and be connected in the water-soluble polymers that is present in the non-genomic coded amino acid in the II zone, site, the hGH polypeptide receptor can not mesh the II zone, site of hGH polypeptide ligand and dimer or polymer and serve as the hGH polypeptide antagonist.In certain embodiments, one or more that are present in the dimer that contains the hGH polypeptide or the hGH molecule in the polymer comprise and are connected in the non-naturally encoded amino acids that is present in the water-soluble polymers in the I calmodulin binding domain CaM of site, thereby allow binding site II zone.Perhaps, in certain embodiments, one or more that are present in the dimer that contains the hGH polypeptide or the hGH molecule in the polymer comprise to be connected in and are present in the not non-naturally encoded amino acids of the water-soluble polymers in site I or site II calmodulin binding domain CaM, thereby it all can be used for combination.In certain embodiments, use has the combination that can be used for bonded site I, site II or both hGH molecules.Wherein at least one have can be used for combination that bonded site I and at least one have the hGH molecule that can be used for bonded site II can provide have the molecule of the activity of wanting or characteristic.In addition, the combination with the hGH molecule that can be used for bonded site I and site II can produce super agonist hGH molecule.

In certain embodiments, GH supergene family member polypeptide (including, but is not limited to) directly connects via Asn-Lys amido linkage or Cys-Cys disulfide linkage.In certain embodiments, GH supergene family member's polypeptide that is connected and/or the non-GH supergene family member who is connected will comprise different non-naturally encoded amino acids and be beneficial to dimerization, and it includes, but is not limited to via Huisgen[3+2] cycloaddition is bonded on the nitrine in the non-naturally encoded amino acids of alkynes in the non-naturally encoded amino acids of a hGH polypeptide and the 2nd GH supergene family member polypeptide.Perhaps, comprise the GH supergene family member's polypeptide that contains the ketone non-naturally encoded amino acids and/or connected comprise the non-GH supergene family member polypeptide that contains the ketone non-naturally encoded amino acids and can be engaged to and comprise the 2nd GH supergene family member polypeptide that contains the azanol non-naturally encoded amino acids, and polypeptide reacts via forming corresponding oxime.

Perhaps, described two GH supergene family member's polypeptide and/or the non-GH supergene family member that connected connect via connexon.Can use any assorted or non-GH supergene family member polypeptide that homotype difunctionality connexon connects described two GH supergene family member's polypeptide and/or connected, it can have identical or different primary sequence.In some cases, be used for the connexon that described GH supergene family member's polypeptide and/or the non-GH supergene family member polypeptide that connected are limited in together be can be the difunctionality polyethylene glycol reagents.

In certain embodiments, the invention provides water-soluble difunctionality connexon, it has the dumbbell-shaped structure that comprises following functional group: a) contain nitrine, alkynes, hydrazine, hydrazides, azanol or carbonyl moiety at least the first end of polymer backbone; And b) at least one second functional group on second end of described polymer backbone.Described second functional group can be identical or different with described first functional group.In certain embodiments, described second functional group does not react with described first functional group.In certain embodiments, the invention provides the water-soluble cpds of the arm that comprises at least one branch molecular structure.For example, described branch molecular structure can be dendritic.

In certain embodiments, the invention provides by reacting one or more the polymers that comprises that form such as the GH supergene family member of hGH with water-soluble activated polymer with following structure:

R-(CH₂CH₂O)_n-O-(CH₂)_m-X

Wherein n is about 5 to 3,000, and m is 2-10, and X can be and contains nitrine, alkynes, hydrazine, hydrazides, aminooxy, azanol, ethanoyl or carbonyl moiety, and R be can be identical or different with X capping group, functional group or leaving group.R can be the functional group who for example is selected from the group that is made up of following group: hydroxyl; hydroxyl and protected; alkoxyl group; N-maloyl imido grpup ester; 1-benzotriazole base ester; carbonic acid N-maloyl imido grpup ester; carbonic acid 1-benzotriazole base ester; acetal; aldehyde; aldehyde hydrate; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; aminooxy; shielded amine; hydrazides; shielded hydrazides; shielded mercaptan; carboxylic acid; shielded carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulphone; two thiopyridines; vinyl pyridine; iodo-acid amide; epoxide; oxalic dialdehyde; diketone; methanesulfonates; tosylate and trifluoro esilate; alkene and ketone.

XII. measuring h GH polypeptide active and hGH are to the avidity of hGH polypeptide receptor

Can be as people such as McFarland, Science, 245:494-499 (1989) and Leung, people such as D., Nature, the described hGH acceptor for preparing of 330:537-543 (1987).Can use in the outer or body of standard body calibrating to measure the hGH polypeptide active.For example, proliferating cells system (for example expressing the clone of hGH acceptor or prolactin acceptor) can be in order to monitoring hGH receptors bind in the presence of hGH.For example, referring to Clark, people such as R., J.Biol.Chem.271 (36): 21969 (1996); People such as Wada, Mol.Endocrinol.12:146-156 (1998); Gout, people such as P.W., Cancer Res.40,2433-2436 (1980); WO 99/03887.For the non-Pegylation hGH polypeptide that comprises alpha-non-natural amino acid, described hormone can be by using BIAcore to the avidity of its acceptor^TMBiosensor (Pharmacia) is measured.For example referring to United States Patent (USP) the 5th, 849, No. 535; Spencer, people such as S.A., J.Biol.Chem., 263:7862-7867 (1988).Be used to test the interior animal model of the active body of hGH and comprise that those for example are described in people such as Clark, the animal model among J.Biol.Chem.271 (36): the 21969-21977 (1996).Calibrating to the dimerization ability of the hGH polypeptide that comprises one or more non-naturally encoded amino acids can be as Cunningham, B. wait the people, Science, 254:821-825 (1991) and Fuh, G. wait people Science, described the carrying out of 256:1677-1680 (1992).The reference and the patent of all references all are incorporated herein by reference.

More than to the compilation of the reference of analytical procedure and non-exhaustive, and it will be understood by one of ordinary skill in the art that the analysis that other is applicable to the test end results of wanting.

XIII. measure transformation period and pharmacokinetic parameter in usefulness, the functive

An importance of the present invention is by will or the hGH polypeptide not being engaged the biological half-life of the prolongation of the described polypeptide acquisition of water-soluble polymers section construction.The quick reduction of hGH polypeptide serum-concentration makes assessment to becoming important with the biological respinse that engages or do not engage the processing of hGH polypeptide and varient thereof.Preferably, joint of the present invention or unassembled hGH polypeptide and varient thereof also have the serum half-life of prolongation after the i.v. dispensing, and making may be by for example ELISA method or by department level screening analysis to measure.Can use from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, ELISA TX) or RIA test kit.The measurement of carrying out biological half-life in the body as described herein.

Comprising in the usefulness of hGH polypeptide of non-naturally encoded amino acids and the functive transformation period can be according to being described in people J.Biol.Chem.271 such as Clark, R., and 36, the scheme among the 21969-21977 (1996) is measured.

The pharmacokinetic parameter that comprises the hGH polypeptide of non-naturally encoded amino acids can assessment in normal Sprague-Dawley male rat (N=4 animal in every treatment group).Animals received single dose 25 micrograms/rat iv or 50 micrograms/rat sc, and take about 5-7 blood sample according to scheduled time process, described time course is for comprising that hGH polypeptide that non-naturally encoded amino acids is not engaged in water-soluble polymers generally included about 6 hours and generally include about 4 days for the hGH polypeptide that comprises non-naturally encoded amino acids and be engaged in water-soluble polymers.The pharmacokinetic data of hGH polypeptide is fully studied in some species and can directly directly be compared with the data that the hGH polypeptide that comprises non-naturally encoded amino acids is obtained.For the research relevant with hGH referring to people Pharm.Res.8 (11): 1351-59 (1991) such as Mordenti J..

Specific activity according to hGH polypeptide of the present invention can be by known various assay determinations in the affiliated field.The hGH polypeptide mutain of acquisition and purifying or its segmental biological activity can be by described herein or reference or the known method tests of those skilled in the art according to the present invention.

XIV. offer medicine and medical composition

Polypeptide of the present invention or albumen (include, but is not limited to hGH, synthetic enzyme, comprise the albumen of one or more alpha-non-natural amino acids etc.) are used for the treatment of purposes according to circumstances, include, but is not limited to be used in combination with suitable medical supporting agent.These compositions for example comprise compound and the pharmaceutically acceptable supporting agent or the vehicle for the treatment of significant quantity.Described supporting agent or vehicle include, but is not limited to salt solution, buffer saline, dextrose, water, glycerine, ethanol and/or its combination.The preparation composite is to be fit to the dispensing pattern.Usually throw and give proteic method and be well-known in affiliated field and can be used for throwing and give polypeptide of the present invention.

The therapeutic composition that comprises one or more polypeptide of the present invention is tested in the animal model in the external and/or body of one or more suitable diseases to confirm effect, tissue metabolism and to assess dosage according to well-known method in the affiliated field according to circumstances.Particularly, dosage just begins by relatively the non-natural of this paper and activity, stability or other suitable measurement (promptly in correlation analysis) of natural amino acid homologue (including, but is not limited to compare with hGH polypeptide and the natural amino acid hGH polypeptide that comprises one or more alpha-non-natural amino acids modified) are determined.

Dispensing is by being generally used for introducing molecule with final contact blood or histiocytic any paths.Non-natural amino acid polypeptides of the present invention is to throw with one or more pharmaceutically acceptable supporting agents in any appropriate manner, according to circumstances to give.Have now the described polypeptide in the literary composition of the present invention is thrown the proper method that gives the patient, although and can use more than one paths to throw and give particular composition, particular path can provide than more timely and more effective effect in another path or reaction.

The particular composition that pharmaceutically acceptable supporting agent part is given by throwing and determine by being used to throw the ad hoc approach that gives described composition.Therefore, the suitable composite that has multiple medical composition of the present invention.

Peptide composition can be oral by including, but is not limited to, intravenously, intraperitoneal, intramuscular, throw through the several paths of skin, subcutaneous, local, hypogloeeis or rectum method and to give.The composition that comprises the non-natural amino acid polypeptides of modification or unmodified also can give via the liposome throwing.Described dosing way and suitable composite are known to the those skilled in the art usually.

Also can be prepared as via sucking separately or with the hGH polypeptide that comprises alpha-non-natural amino acid of other suitable component combination and to throw the aerosol composite (being that it can " atomize ") that gives.The aerosol composite can place the propelling agent accepted such asRefrigerant 12, propane, nitrogen or the like of pressurization.

Be applicable to such as by intraarticular (in the joint), intravenously, intramuscular, intracutaneous, intraperitoneal and subcutaneous route non-through intestines throw and composite comprise the water-based and an aseptic injectable solution such as non-aqueous of the solute that the blood that can contain antioxidant, buffer reagent, fungistat and make described composite and predetermined receptor etc. is opened and can comprise the water-based and the non-aqueous sterile suspensions of suspension agent, solubilizing agent, thickening material, stablizer and sanitas.In the composite of nucleic acid of packing can be present in unitary dose or multiple doses sealed vessel such as ampoule and bottle.

Non-is preferred medication administration method through intestines dispensing and intravenously dispensing.Specific, the dosing way (including but not limited to be generally used for EPO, GH, G-CSF, GM-CSF, IFN, interleukin, antibody and/or any proteic dosing way that other pharmaceutically transmits) that has been used for natural amino acid homologue therapeutical agent is provided for preferred throwing and the approach and the composite of polypeptide of the present invention with the composite of current use.

Throwing dosage with the patient in the context of the present invention is enough in the patient to have useful therapeutic response or to include but not limited to be enough to suppress pathogenic infection or look to use along with the time other suitable active is provided.Described dosage is to be determined by the patient's of the activity of the non-natural amino acid polypeptides of the effect of specific support or composite and application, stability or serum half-life and status of patient and desire treatment body weight or surface-area.

The size of dosage also by the throwing of following specific support, composite or the like in the particular patient and the existence, nature and extent of any adverse side effect determine.In determining treatment or preventing disease (including but not limited to cancer, heredopathia, diabetes, AIDS or the like) the desire throwing and carrier or during the significant quantity of composite, doctor's assessments plasma content, composite toxicity, disease process and/or anti-non-natural amino acid polypeptides production of antibodies have the dependency part.

The dosage of throwing and for example 70 kg of patient in the scope that is equivalent to the proteic dosage of currently used treatment, is regulated according to the activity or the serum half-life of compositions related change usually.Carrier of the present invention can any known routine treatment supplement therapy condition, described routine treatment include but not limited to antibody throw with, vaccine is thrown and, throwing and cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, biological response modifier or the like.

For throw with, with by the LD-50 or the ED-50 of relative allocation thing and/or include but not limited to be applied to the observation of any side effect of alpha-non-natural amino acid under the various concentration of patient's quality and general health and definite speed is thrown and composite of the present invention.Throw and can finish via dosage single or that separate.

If the patient of experience composite infusion heating occurs, feels cold or myalgia, then he receives acetylsalicylic acid (aspirin), Ibuprofen BP/EP (ibuprofen), acetaminophen (acetaminophen) or other pain/heating control medicine of suitable dosage.Experience used acetylsalicylic acid, acetaminophen or (including but not limited to) diphenhydramine (diphenhydramine) to treat the patient of the reaction of infusion (such as heating, myalgia and feel cold) in preceding 30 minutes at the infusion in future in advance.Pethidine (meperidine) is used for febrifuge and not more serious the feeling cold and myalgia of reaction rapidly of antihistaminic agent.The seriousness of looking described reaction is slowed down or is interrupted cell infusion.

Human hGH polypeptide of the present invention can directly be thrown and the Mammals person under inspection.Throw and be by being generally used for the hGH polypeptide is introduced any approach of person under inspection.According to an embodiment of the invention the hGH peptide composition comprise be suitable for oral, rectum, part, suction (including but not limited to), oral cavity (including but not limited to the hypogloeeis), vagina via aerosol, non-(be skin and mucomembranous surface through intestines (include but not limited in subcutaneous, intramuscular, intracutaneous, intraarticular, the pleura, in the intraperitoneal, brain, intra-arterial or intravenously), part, comprise the tracheae surface) and through skin throwing and person, although the optimum approach in any given situation will depend on the character and the seriousness of the illness of desire treatment.Throw and can be part or general.The composite of compound can be present in such as in the unitary dose of ampoule and bottle or the multiple doses sealed vessel.

HGH polypeptide of the present invention can be prepared as the mixture of unitary dose injectable forms (including but not limited to solution, suspension or emulsion) and pharmaceutically acceptable supporting agent.HGH polypeptide of the present invention also can by formula injection of continuous infusion (use includes but not limited to the micropump such as osmotic pump), single group or slow releasing pharmaceutical storage tank composite throw with.Be suitable for throwing and composite comprise and can contain antioxidant, buffer reagent, fungistat and make the water-based of the solute that described composite etc. is opened and non-aqueous solution etc. open sterile solution and can comprise the water-based and the non-aqueous sterile suspensions of suspension agent, solubilizing agent, thickening material, stablizer and sanitas.Solution and suspension can be from sterilized powder, particle and the pharmaceutical tablet of previous described kind.Medical composition of the present invention can comprise pharmaceutically acceptable supporting agent.

Pharmaceutically acceptable supporting agent partly be by throw and particular composition and determine by the ad hoc approach that is used to throw with described composition.Therefore, there is the multiple medical composition of the present invention suitable composite (for example referring to Remington ' s Pharmaceutical Sciences, the 17th edition, 1985) of (comprising optionally pharmaceutically acceptable supporting agent, vehicle or stablizer).

Suitable supporting agent comprises and contains phosphoric acid salt, borate, HEPES, Citrate trianion and other organic acid buffer reagent; The antioxidant that comprises xitix; Lower molecular weight (less than about 10 residues) polypeptide; Albumen such as serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer such as polyvinylpyrrolidone; Such as glycine, glutamine, l-asparagine, arginine or lysine amino acid; The monose, disaccharides and other carbohydrate that comprise glucose, seminose or dextrin; Sequestrant such as EDTA; Divalent-metal ion such as zinc, cobalt or copper; Sugar alcohol such as mannitol or Sorbitol Powder; Salify counterion such as sodium; And/or such as Tween^TM, Pluronics^TMOr the nonionogenic tenside of PEG.

Comprise be connected in such as the hGH polypeptide of the present invention of the water-soluble polymers of PEG also can by sustained release system or as the part of sustained release system throw with.Sustained-release composition comprises that (including but not limited to) includes but not limited to the semipermeability polymeric matrix of the setting article form of film or microcapsule.Lasting release matrix comprises biocompatible materials, such as poly-(methacrylic acid 2-hydroxyl ethyl ester) (people such as Langer, J.Biomed.Mater.Res., 15:167-277 (1981); Langer, Chem.Tech., 12:98-105 (1982)), ethylene vinyl acetate (people such as Langer, the same) or poly--(EP 133 for D-(-)-3-hydroxybutyric acid, 988), polylactide (poly(lactic acid)) (United States Patent (USP) the 3rd, 773, No. 919, EP58,481), poly-glycollide (polymkeric substance of oxyacetic acid), polylactide-altogether-glycollide (multipolymer of lactic acid and oxyacetic acid), polyanhydride, the multipolymer of L-L-glutamic acid and γ-ethyl-L-glutaminate (people such as U.Sidman, Biopolymers, 22,547-556 (1983)), poly-(ortho acid) ester, polypeptide, hyaluronic acid, collagen protein, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, nucleic acid, polyamino acid, such as phenylalanine, tyrosine, the amino acid of Isoleucine, polynucleotide, polyethylene propylene, polyvinylpyrrolidone and siloxanes.Sustained-release composition also comprises the compound of liposome encapsulation.The liposome that contains described compound comes by known method preparation own: DE 3,218, and 121; People such as Epstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485,045 and 4,544, No. 545 and EP102,324.The reference and the patent of all references all are incorporated herein by reference.

The hGH polypeptide that captures by liposome can prepare by the described method of following document: for example, and DE3,218,121; People such as Epstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl.Acad.Sci.U.S.A., 11:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485,045 and 4,544, No. 545; And EP 102,324.The composition of liposome and size are that well-known maybe can being easy to rule of thumb come to determine by the those skilled in the art.Some case descriptions of liposome are in following document: for example, and people such as Park JW, Proc.Natl.Acad.Sci.USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (volume): MedicalApplications of Liposomes (1998); People such as Drummond DC, Liposomal drug delivery systemsfor cancer therapy, Teicher B (volume): CANCER Drug Discovery and Development (2002); People such as ParkJW, Clin.Cancer Res.8:1172-1181 (2002); People such as Nielsen UB, Biochem.Biophys.Acta1591 (1-3): 109-118 (2002); People such as Mamot C, Cancer Res.63:3154-3161 (2003).All documents and the patent of being quoted all are incorporated herein by reference.

In the context of the present invention, the dosage that comes into operation to the patient should be enough to cause the time dependent favourable reaction of person under inspection.Usually, total the non-medicine and pharmacology significant quantity through the hGH polypeptide of the present invention that intestines come into operation of each dosage about 0.01 μ g/kg/ days to about 100 μ g/kg or about 0.05mg/kg to the scope of about 1mg/kg weight in patients, although this is easy to be arranged by the treatment judgement.The frequency of administration also is easy to judge domination by treatment, and comparable approval to be used for human commercially available hGH polypeptide products more frequent or more rare.Usually, Pegylation hGH polypeptide of the present invention can be offerd medicine by in the above-mentioned dosing way any.

The therepic use of hGH polypeptide XV. of the present invention

HGH polypeptide of the present invention is applicable to the various illnesss of treatment.

For example, hGH agonist polypeptide of the present invention is applicable to treating growth defect, immune disorders and being applicable to the cardiac stimulus function.The individuality of suffering from growth defect comprises: for example, suffer from the individuality of Tener syndromes; GH defective individuality (comprising children); Children's (being called " microsomia children " sometimes) that about 2-3 experience normal growth curve slows down or postpones before the growth plate closure; And wherein insulin-like growth factor-I (IGF-I) reaction of GH is passed through chemical mode (for example, pass through glucocorticoid treatment) or by natural condition (for example, in adult patients, natural the slowing down of IGF-I reaction of wherein GH being made) individuality of blocking-up.

Agonist hGH varient can work to stimulate immune system by increasing immunologic function, no matter whether causes described increase because antibody is regulated or cell is regulated and no matter immunity system is for whether being endogenic for the host of hGH polypeptide treatment or not transplanted to the host receptor of accepting the hGH polypeptide (as in the bone marrow transplantation body) by donor." immune disorders " comprises any illness that individual immunity system wherein reduces than the reaction of normal individual (comprise spleniculus have the immunity of minimizing because of medicine (chemotherapy for example) treatment those individualities) to some extent to antigenic antibody or cell response.The example individuality of suffering from immune disorders comprises: for example, and the gerontal patient; Accept chemotherapy or radiotherapeutic individuality; Restore the individuality that maybe will undergo surgery from grave illness; The individuality of suffering from AIDS; Suffers from for example patient of the congenital and acquired character B cell defect of hypogammaglobulinemia, the no gamma-globulin mass formed by blood stasis (common varied agammaglobulinemia) that changes usually and selective immunoglobulin deficiency (IgG defective for example); Infect for example patient of rabic virus, the incubation time of described virus is shorter than patient's immune response; And suffers from for example individuality of the hereditary illness of enlightening George syndromes.

HGH antagonist polypeptide of the present invention is applicable to treatment gigantosoma and the reactive malignant tumour of acromegaly, diabetes and the complication (diabetic retinopathy, diabetic neuropathy) that is caused by diabetes, blood vessel illness in eye (for example, relating to the proliferative neovascularization), ephrosis and GH.

For example, blood vessel illness in eye comprises retinopathy (for example, being caused by precocity or sicklemia) and macular degeneration.

For example, the reactive malignant tumour of GH comprises: the cancer of the tissue of Weir Mu Shi tumour, sarcoma (osteogenic sarcoma for example), breast cancer, colorectal carcinoma, prostate cancer and thyroid carcinoma, expression GH receptor mrna (be placenta, thymus gland, brain, sialisterium, prostate gland, marrow, skeletal muscle, backbone, spinal cord, retina, lymphoglandula and from Bark lymphomas, colorectal carcinoma, lung cancer, lymphoid leukemia and melanomatous tissue).

The average quantity of hGH can change and especially should be based on the recommendation and the prescription of qualified physicians.The accurate amount of hGH is the problem of a different people, different views, is arranged by the accurate type of for example symptom for the treatment of, the patient's that treats healthy state and the factor of other composition in the composition.

Example

Provide following example with explanation rather than restriction the present invention.

Example 1

This case description is used for selecting non-naturally encoded amino acids is incorporated into one of the many groups of potential standards of the preferred sites of hGH.

This example confirms how to select preferred sites in the hGH polypeptide to introduce non-naturally encoded amino acids.Use comprises the optimum position of determining wherein can introduce one or more non-naturally encoded amino acids with the crystalline structure 3HHR of two molecule compound hGH of acceptor (hGHbp) cell foreign lands.Other hGH structure (for example 1AXI) is to be used for detecting the one-level between the crystalline structure data set and the potential variation of secondary building unit.The coordinate of these structures can derive from albumen data bank (PDB) (people such as Berstein, J.Mol Biol 1997,112, the 535 pages) or can derive from World Wide Web rcsb.org via The ResearchCollaborator for Structural Bioinformatics PDB.Structural models 3HHR contains the complete ripe 22kDa sequence of hGH, but in the crystal because of except unordered elliptical residue 148-153 and the C-terminal F191 residue.Two disulphide bridgeses that existence is formed by C53 and C165 and C182 and C185.The sequence numbering that is used for this example is the aminoacid sequence according to the ripe hGH (22kDa varient) shown in the SEQ ID NO:2.

Following standard is each position that is used to estimate the hGH that is used to introduce non-naturally encoded amino acids: residue (a) should not disturb combining based on arbitrary hGHbp of the structural analysis of 3HHR, 1AXI and 1HWG (crystalline texture of the hGH that engages with hGHbp monomer or dimer); (b) not brought out by the scanning sudden change of L-Ala or homologue influences people such as (, people such as Science (1989) 244:1081-1085 and Cummingham, Science (1989) 243:1330-1336) Cunningham; (c) should the surface expose and show and the minimum Van der Waals or the interaction of hydrogen bond of residue on every side; (d) in hGH varient (for example Tyr35, Lys38, Phe92, Lys140), should omit maybe and can change; (e) after non-naturally encoded amino acids replaces with being about to cause conservative change; And (f) be found in high degree of flexibility zone (include, but is not limited to CD ring) or structure rigidity zone (the including, but is not limited to spiral B).In addition, on the hGH molecule, carry out other calculating, utilize Cx program people such as (, Bioinformatics, 18, the 980 pages) Pintar to estimate the projecting degree of each albumen atom.The result, in certain embodiments, one or more non-naturally encoded amino acids are incorporated into one or more positions in the following column position of hGH: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).

In certain embodiments, one or more non-naturally encoded amino acids one or more positions in following column position are substituted: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).

In certain embodiments, one or more non-naturally encoded amino acids one or more positions in following column position are substituted: 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186 and 187 (SEQ ID NO:2, or the corresponding amino acid in SEQID NO:1 or 3).

In certain embodiments, one or more non-naturally encoded amino acids in following column position one or individual above position are substituted: 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).

In certain embodiments, one or more non-naturally encoded amino acids one or more positions in following column position are substituted: 30,74,103 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).In certain embodiments, one or more non-naturally encoded amino acids one or more positions in following column position are substituted: 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).

In certain embodiments, the non-natural of one or more positions in these positions exists amino acid to be connected in water-soluble polymers, described position includes, but is not limited to down column position: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (being proteic C-terminal) (SEQID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).In certain embodiments, the non-natural of one or more positions in these positions exists amino acid to be connected in water-soluble polymers: 30,35,74,92,103,143,145 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).In certain embodiments, the non-natural of one or more positions in these positions exists amino acid to be connected in water-soluble polymers: 35,92,143,145 (SEQ ID NO:2, or the corresponding amino acid in SEQ ID NO:1 or 3).

Some sites that produce the hGH antagonist comprise: 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123,127 orposition 1 before interpolation or any its combination (SEQ ID NO:2, or the corresponding amino acid among the SEQ ID NO:1,3, or any other GH sequence).Utilize the standard (c)-(e) of agonist design to select these sites.The antagonist design can comprise that also the pointed decoration of site I residue is with the binding affinity of increase with hGHbp.

Example 2

This example has described the hGH polypeptide that in intestinal bacteria (E.coli) cloning and expression comprises non-naturally encoded amino acids in detail.This example is also described a kind of bioactive method of assessing modified hGH polypeptide.

Be used to clone hGH and segmental method is specified in United States Patent (USP) the 4th, 601,980,4,604,359,4,634,677,4,658,021,4,898,830,5,424,199 and 5,795, in No. 745, described patent is incorporated herein by reference.The cDNA of the hGH mature form of coding total length hGH or shortage N-terminal signal sequence is shown in respectively among SEQ ID NO:21 and the SEQ ID NO:22.

The translation system of being introduced that comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) is to be used to express the hGH that contains non-naturally encoded amino acids.O-RS favors in using non-naturally encoded amino acids aminoacylation O-tRNA.Described translation system then reply coded selection codon non-naturally encoded amino acids is inserted among the hGH.

Table 2:O-RS and O-tRNA sequence

SEQ?ID?NO：4	Methanobacteria (M.jannaschii) mtRNATyrCUA	tRNA
			SEQ?ID?NO：5	HLAD03; Best amber suppresses sub-tRNA	tRNA
SEQ?ID?NO：6	HL325A; Best AGGA frameshit suppresses sub-tRNA	tRNA
			SEQ?ID?NO：7	Be used to incorporate into aminoacyl tRNA synthetase to azido--L-phenylalanine p-Az-PheRS (6)	RS
SEQ?ID?NO：8	Be used to incorporate into aminoacyl tRNA synthetase to benzoyl-L-phenylalanine p-BpaRS (l)	RS
			SEQ?ID?NO：9	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine propargyl-PheRS	RS
SEQ?ID?NO：10	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine propargyl-PheRS	RS
			SEQ?ID?NO：11	Be used to incorporate into the aminoacyl tRNA synthetase of propargyl-phenylalanine propargyl-PheRS	RS
SEQ?ID?NO：12	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine p-Az-PheRS (l)	RS
			SEQ?ID?NO：13	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine p-Az-PheRS (3)	RS
SEQ?ID?NO：14	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine p-Az-PheRS (4)	RS
			SEQ?ID?NO：15	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine p-Az-PheRS (2)	RS
SEQ?ID?NO：16	Be used to incorporate into aminoacyl tRNA synthetase to ethanoyl-phenylalanine (LW1)	RS
			SEQ?ID?NO：17	Be used to incorporate into aminoacyl tRNA synthetase to ethanoyl-phenylalanine (LW5)	RS
SEQ?ID?NO：18	Be used to incorporate into aminoacyl tRNA synthetase to ethanoyl-phenylalanine (LW6)	RS
			SEQ?ID?NO：19	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine (AzPheRS-5)	RS
SEQ?ID?NO：20	Be used to incorporate into aminoacyl tRNA synthetase to azido--phenylalanine (AzPheRS-6)	RS

Non-naturally encoded amino acids can be incorporated in the hGH polypeptide with locus specificity the plasmid transformation escherichia coli of (want non-naturally encoded amino acids is had specificity) with containing modified hGH gene and quadrature aminoacyl tRNA synthetase/tRNA.Intestinal bacteria through transforming are being contained in the substratum of the specific non-naturally encoded amino acids of 0.01-100mM 37 ℃ of growths down, and described escherichia coli expression has the modified hGH of high frequency high fidelity and efficient.The hGH through the His mark that Bacillus coli cells will contain non-naturally encoded amino acids is produced as inclusion body or aggregate.Under the sex change condition, described aggregate is dissolved in the 6MHCl guanidine and carry out affinity purification.At 50mM TRIS-HCl (pH 8.0), 40 μ MCuSO₄And among 2% (w/v) Sarkosyl, under 4 ℃, carry out again folding by dialysed overnight.Then, make described material by 20mM TRIS-HCl (pH 8.0), 100mM NaCl, 2mM CaCl₂Dialyse, remove the His mark then.See people such as Boissel, (1993) 268:15983-93.The method that is used for purifying hGH is well-known in affiliated field and is analyzed or electron spray(ES)-ionization ion trap mass spectrometry and similar approach thereof confirm by SDS-PAGE, Western Blot.

Fig. 6 is the SDS-PAGE of purified hGH polypeptide.Use ProBond Nickel-Chelating Resin (Invitrogen via the standard that manufacturers provides through His labelled protein purifying procedure, Carlsbad, CA), before being carried on the gel, come purifying through the mutant hGH of His mark albumen then via the anionresin tubing string.The N-His hGH of alpha-non-natural amino acid is not incorporated inswimming lane 1 demonstration molecular weight marker andswimming lane 2 expressions into.Swimming lane 3-10 contains respectively, and the everywhere in position Y35, F92, Y111, G131, R134, K140, Y143 and K145 comprises the N-His hGH mutant of alpha-non-natural amino acid to ethanoyl-phenylalanine.

For further analyzing the biological activity of modified hGH polypeptide, use the downstream mark of measuring h GH and the interactional calibrating of its acceptor.The interaction of the acceptor of hGH generation endogenous with it causes the signal transducer of transcribing family member STAT5 in the human IM-9 lymphocyte series and the tyrosine phosphorylation of activator.Two kinds of forms by IM-9cDNA storehouse identification STAT5, STAT5A and STAT5B.For example, see people such as Silva, Mol.Endocrinol. (1996) 10 (5): 508-518.Human growth hormone's Receptor in Human class tethelin on the IM-9 cell has selectivity, because rat growth hormone or human prolactin antagonist all can not cause detectable STAT5 phosphorylation.Importantly, rat GHR (L43R) cell foreign lands and have the pSTAT5 phosphorylation that the hGH effective competition antagonism of G120R stimulates through hGH.

Stimulate the IM-9 cell with hGH polypeptide of the present invention.Human IM-9 lymphocyte is available from ATCC (Manassas, VA) and grow in and be supplemented with Sodium.alpha.-ketopropionate, penicillin, Streptomycin sulphate (Invitrogen, Carlsbad, San Diego) and 10% heat-inactivated fetal bovine serum (Hyclone, Logan is among the RPMI 1640 UT).Before 37 ℃ of following hGH polypeptide with 12 dose point scopes stimulate 10 minutes, make IM-9 cell (no phenol red RPMI, 10mM Hepes, FBS, Sodium.alpha.-ketopropionate, penicillin and the Streptomycin sulphate that 1% hot deactivation charcoal/dextran is handled) hunger of spending the night in the calibrating substratum.Before permeating 1 hour on ice, with the cell of 1% formaldehyde fixed irriate with 90% ice cold methanol.(Cell Signaling Technology, Beverly MA) carry out cell inner dyeing, last 30 minutes, the second antibody dyeing that engages with PE then, thereby detection STAT5 phosphorylation degree at room temperature to use first phosphate-STAT5 antibody.Carry out sample and obtain on the FACS array, (Tree Star Inc., Ashland OR) go up analysis and the data of being obtained are at Flowjo software.Draw EC by the dose response curve that utilizes SigmaPlot to draw with average fluorescent strength (MFI)-protein concentration₅₀Value.

Following table 3 has been summarized the IM-9 data that produced by mutant hGH polypeptide.Test the various hGH polypeptide that have the alpha-non-natural amino acid replacement at different positions with aforesaid human IM-9 cell.Particularly, Fig. 7 picture A shows the IM-9 data through the hGH of His mark polypeptide, and Fig. 7 picture B shows and comprises the alpha-non-natural amino acid that the replaces Y143 IM-9 data through the hGH of His mark to ethanoyl-phenylalanine replacement.The biological activity of the hGH polypeptide that uses identical calibrating to assess to comprise the Pegylation alpha-non-natural amino acid.

Table 3
					GH	EC50(nM)		GH	EC50(nM)
WHOWT	0.4±0.1(n＝8)		G120R	＞200,000
					N-6His?WT	0.6±0.3(n＝3)		G120pAF	＞200,000
Rat GH WT	＞200,000		G131pAF	0.8±0.5(n＝3)
					Y35pAF	0.7±0.2(n＝4)		P133pAF	1.0
E88pAF	0.9		R134pAF	0.9±0.3(n＝4)
					Q91pAF	2.0±0.6(n＝2)		T135pAF	0.9
F92pAF	0.8±0.4(n＝9)		G136pAF	1.4
					R94pAF	0.7		F139pAF	3.3
S95pAF	16.7±1.0(n＝2)		K140pAF	2.7±0.9(n＝2)
					N99pAF	8.5		Y143pAF	0.8±0.3(n＝3)
Y103pAF	130,000		K145pAF	0.6±0.2(n＝3)
					Y111pAF	1.0		A155pAF	1.3

Example 3

This example described in detail the amino acid whose introducing that contains carbonyl and with the subsequent reactions of the PEG that contains aminooxy.

This example confirms a kind of method that is used to produce the hGH polypeptide, incorporates subsequently with about 5 the non-naturally encoded amino acids that contains ketone of the PEG reaction that contains aminooxy of 000MW in the described polypeptide into.Each independent non-naturally encoded amino acids through having following array structure in the residue of discerning according to the standard of example 1 (hGH) 35,88,91,92,94,95,99,101,103,111,120,131,133,134,135,136,139,140,143,145 and 155 is replaced:

Being used for the sequence that the locus specificity to ethanoyl-phenylalanine of hGH incorporates into is SEQ ID NO:2 (hGH) and SEQ ID NO:4 (muttRNA, methanobacteria mtRNA^Tyr_CUA) and above example 2 in 16,17 or 18 (the TyrRS LW1,5 or 6) that describe.

In case modified, comprise contain carbonyl amino acid whose hGH polypeptide variants will with the PEG derivatives reaction that contains aminooxy of following form:

R-PEG(N)-O-(CH₂)_n-O-NH₂

Wherein R is a methyl, n be 3 and N be about 5,000MW.Make to contain and be dissolved in 25mM MES (Sigma Chemical with 10mg/mL; St.Louis; MO) (pH 6.0), 25mM Hepes (Sigma Chemical; St.Louis; MO) (pH 7.0) or 10mM sodium acetate (Sigma Chemical; St.Louis; MO) purified hGH and the doubly excessive PEG reaction that contains aminooxy of 10-100 in (pH 4.5) to acetyl phenyl alanine; and at room temperature stir 10-16 hour (Jencks then; W.J.Am.Chem.Soc.1959; 81, the 475 pages).Then, PEG-hGH is diluted in the suitable damping fluid with purifying and analysis immediately.

Example 4

Engage with the PEG that forms by the azanol base that is connected in PEG via amido linkage.

The program of describing in the use-case 3 makes PEG reagent coupling with following array structure in the non-naturally encoded amino acids that contains ketone:

R-PEG(N)-O-(CH₂)₂-NH-C(O)(CH₂)_n-O-NH₂

R=methyl wherein, n=4 and N are about 20,000MW.Described reaction, purifying and analysis condition are as described in the example 3.

Example 5

This example has described in detail to be introduced two kinds of different non-naturally encoded amino acids in the hGH polypeptide.

This example confirms a kind of method that is used to produce the hGH polypeptide, and two positions in the described polypeptide in following residue are incorporated the non-naturally encoded amino acids that comprises the ketone functional group: E30, E74, Y103, K38, K41, K140 and K145 into.As described in example 1 and 2, prepare the hGH polypeptide, but two different loci in nucleic acid are introduced except the inhibition codon.

Example 6

This example has described in detail and the hGH polypeptide has been engaged and in-situ reducing subsequently with the PEG that contains hydrazides.

Prepare according to example 2 and 3 described programs and to incorporate the amino acid whose hGH polypeptide that contains carbonyl into.In case modified, the PEG that contains hydrazides with following array structure is engaged with the hGH polypeptide:

R-PEG(N)-O-(CH₂)₂-NH-C(O)(CH₂)_n-X-NH-NH₂

R=methyl wherein, n=2 and N=10,000MW and X are carbonyl (C=O).The hGH that will contain acetyl phenyl alanine is dissolved in 25mM MES (Sigma Chemical with 0.1-10mg/mL; St.Louis; MO) (pH 6.0), 25mM Hepes (Sigma Chemical; St.Louis; MO) (pH 7.0) or 10mM sodium acetate (Sigma Chemical, St.Louis is MO) in (pH 4.5); make the doubly excessive PEG reaction that contains hydrazides of itself and 1-100, and by adding 1M NaCNBH₃(Sigma Chemical, St.Louis MO) make corresponding hydrazone in-situ reducing to storing solution, make it be dissolved in H₂Among the O, up to the ultimate density of 10-50mM.In the dark, to room temperature, react, last 18-24 hour at 4 ℃.(Sigma Chemical, St.Louis is MO) to the final Tris concentration of 50mM or be diluted in and be used for immediately that the suitable damping fluid of purifying comes termination reaction for 1M Tris by adding about pH 7.6.

Example 7

This example has described in detail in the amino acid introducing hGH polypeptide that will contain alkynes and with the mPEG-trinitride derives.

Following residue 35,88,91,92,94,95,99,101,131,133,134,135,136,140,143, the 145 and 155 following non-naturally encoded amino acids (hGH that respectively hangs oneself; SEQ ID NO:2) replace:

Being used for the sequence that the locus specificity to propargyl-tyrosine of hGH incorporates into is SEQ ID NO:2 (hGH) and SEQ ID NO:4 (muttRNA, methanobacteria mtRNA^Tyr_CUA) and above example 2 in describe 9,10 or 11.The condition of describing in the use-case 3 makes the hGH expression of polypeptides that contains propargyl tyrosine in intestinal bacteria and carry out purifying.

Make the purified hGH that contains propargyl tyrosine with 0.1-10mg/mL be dissolved in the PB damping fluid (the 100mM sodium phosphate, 0.15M NaCl, pH=8) in and the PEG that contains nitrine that 10-1000 is doubly excessive add in the reaction mixture.Then, with the CuSO of catalytic amount₄Add in the reaction mixture with the Cu line.Afterwards, add H cultivating described mixture (include, but is not limited to room temperature or 37 ℃ about 4 hours down, or under 4 ℃, spend the night)₂O and filter described mixture by dialysis membrane.Can analyze the sample that is added by the similar program of describing in (including, but are not limited to) example 3.

In this example, PEG will have following array structure:

R-PEG(N)-O-(CH₂)₂-NH-C(O)(CH₂)_n-N₃

Wherein R is a methyl, n be 4 and N be 10,000MW.

Example 8

This example has described in detail with big, hydrophobic amino acid in the propargyl tyrosine replacement hGH polypeptide.

Replace Phe among one in the following column region that is present in hGH with the following non-naturally encoded amino acids of describing in the example 7, Trp or Tyr residue: 1-5 (N-terminal), 6-33 (A spiral), 34-74 (the zone between A spiral and the B spiral, the A-B ring), 75-96 (B spiral), 97-105 (the zone between B spiral and the C spiral, the B-C ring), 106-129 (C spiral), 130-153 (the zone between C spiral and the D spiral, the C-D ring), 154-183 (D spiral), 184-191 (C-terminal) (SEQ ID NO:2):

In case modified, PEG promptly is connected in and comprises the amino acid whose hGH polypeptide variants that contains alkynes.Described PEG will have following array structure:

Me-PEG(N)-O-(CH₂)₂-N₃

And the coupling program will be followed the program in the example 7.This comprises the hGH polypeptide variants of non-naturally encoded amino acids with generation, its roughly with naturally occurring big, hydrophobic amino acid in rows such as one and the different loci in polypeptide modify through the PEG derivative.

Example 9

This example has described the generation of the hGH polypeptide homodimer, heterodimer, homopolymer or the heteropolymer that separate by one or more PEG connexons in detail.

Make the hGH polypeptide variants that contains alkynes of generation in the example 7 and the difunctionality PEG derivatives reaction of following form:

N₃-(CH₂)_n-C(O)-NH-(CH₂)₂-O-PEG(N)-O-(CH₂)₂-NH-C(O)-(CH₂)_n-N₃

Wherein n be 4 and PEG have about 5,000 average MW, to produce corresponding hGH polypeptide homodimer, wherein two hGH molecules are by the PEG physical separation.The hGH polypeptide can be in a similar manner with one or more other polypeptide couplings to form heterodimer, homopolymer or heteropolymer.As in example 7 and 3, carry out coupling, purifying and analysis.

Example 10

This example has described the coupling of sugar moieties and hGH polypeptide in detail.

Described in example 3, a residue in the following residue is replaced: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186 and 187 (hGH, SEQ ID NO:2) through non-naturally encoded amino acids hereinafter.

In case modified, make the β that comprises the amino acid whose hGH polypeptide variants that contains carbonyl and N-acetyl glucosamine (GlcNAc)-be connected aminooxy analogue reaction.HGH polypeptide variants (10mg/mL) is mixed in 100mM sodium acetate aqueous buffer solution (pH 5.5) with aminooxy sugar (21mM) and cultivated 7-26 hour down at 37 ℃.At ambient temperature, with UDP-semi-lactosi (16mM) and the p-1 in the 150mM HEPES damping fluid (pH 7.4), (the hGH polypeptide (5mg/mL) that sugar engages is cultivated to the 4-galactosyltransferase by 0.4 unit/mL), last 48 hours, thereby make second sugar and the short coupling of first carbohydrase (people such as Schanbacher, J.Biol.Chem.1970,245,5057-5061).

Example 11

This example has described the generation of Pegylation hGH polypeptide antagonist in detail.

Described in example 3, make followingresidue 1,2,3,4,5,8,9,11,12,15,16,19,22,103,109,112,113,115,116,119,120,123 or 127 (hGH; SEQ ID NO:2, or the corresponding amino acid in SEQID NO:1 or 3) in a residue replace through following non-naturally encoded amino acids.

In case modified, make to comprise the amino acid whose hGH polypeptide variants that contains carbonyl and the PEG derivatives reaction that contains aminooxy of following form:

R-PEG(N)-O-(CH₂)_n-O-NH₂

Wherein R is a methyl, and n is 4 andNWEI 20, and 000MW comprises the hGH polypeptide antagonist of non-naturally encoded amino acids with generation, and its single site in polypeptide is modified through the PEG derivative.As in example 3, carry out coupling, purifying and analysis.

Example 12

Produce hGH polypeptide homodimer, heterodimer, homopolymer or heteropolymer, wherein the hGH molecule directly links to each other.

Comprise contain alkynes amino acid whose hGH polypeptide variants directly coupling another comprise the amino acid whose hGH polypeptide variants that contains azido-, wherein each comprises non-naturally encoded amino acids and replaces in (being not limited to) example 10 described sites.This will produce corresponding hGH polypeptide homodimer, and wherein two hGH polypeptide variants link to each other at II bonding interface place, site physics.The hGH polypeptide can be in a similar manner with one or more other polypeptide couplings to form heterodimer, homopolymer or heteropolymer.As in example 3,6 and 7, carry out coupling, purifying and analysis.

Example 13

A B

Polyalkylene glycol (P-OH) and haloalkane (A) are reacted to form ether (B).In these compounds, n is that integer and the R ' of 1-9 can be straight or branched, saturated or unsaturated C1-C20 alkyl or assorted alkyl.R ' also can be saturated or unsaturated cycloalkyl of C3-C7 or the assorted alkyl of ring, be substituted or unsubstituted aryl or heteroaryl or be substituted or unsubstituted alkaryl (alkyl is the saturated or unsaturated alkyl of C1-C20) or assorted alkaryl.Generally, PEG-OH is that molecular weight is 800-40,000 dalton's (Da) polyoxyethylene glycol (PEG) or mono methoxy polyethylene glycol (mPEG).

Example 14

Making molecular weight is 20,000Da (mPEG-OH 20kDa; 2.0g, 0.1mmol, the NaH among mPEG-OH Sunbio) and the THF (35mL) (12mg, 0.5mmol) reaction.Then, be dissolved in become in the dimethylbenzene propargyl bromination thing of 80 weight % solution solution (0.56mL, 5mmol, 50 equivalents, Aldrich) and the KI of catalytic amount add in the described solution, and with the gained mixture heating up to refluxing, last 2 hours.Then, add water (1mL) and remove solvent in a vacuum.In resistates, add CH₂Cl₂(25mL) and separate organic layer, with anhydrous Na₂SO₄Drying, and volume is reduced to about 2mL.With this CH₂Cl₂Solution dropwise adds in the ether (150mL).Collect the gained throw out, with several parts of cold diethyl ether washings and dry so that propargyl-O-PEG to be provided.

Example 15

With the NaH among the THF (35mL) (12mg, 0.5mmol) handling molecular weight is 20,000Da (mPEG-OH20kDa; 2.0g, 0.1mmol, mPEG-OH Sunbio).Then, (Aldrich) KI with catalytic amount adds in the described mixture for 0.53mL, 5mmol with 50 normal 5-bromo-1-pentynes.The gained mixture heating up to refluxing, is lasted 16 hours.Then, add water (1mL) and remove solvent in a vacuum.In resistates, add CH₂Cl₂(25mL) and separate organic layer, with anhydrous Na₂SO₄Drying, and volume is reduced to about 2mL.With this CH₂Cl₂Solution dropwise adds in the ether (150mL).Collect the gained throw out, with several parts of cold diethyl ether washings and dry so that corresponding alkynes to be provided.5-chloro-1-pentyne can be used in the similar reaction.

Example 16

(1)

(2)

(3)

(4)

At first, (2.4g 20mmol) is dissolved in the solution in THF (50mL) and the water (2.5mL) and adds Powdered sodium hydroxide (1.5g to the 3-salicylic alcohol, 37.5mmol), and add the solution of propargyl bromination thing then, it is dissolved in dimethylbenzene, and (3.36mL becomes 80% solution in 30mmol).Reaction mixture was heated 6 hours under refluxing.In described mixture, add 10% citric acid (2.5mL) and remove solvent in a vacuum.(3 * 15mL) extracted residues and with saturated NaCl solution (10mL) washing combination organic layer are used MgSO with ethyl acetate₄Dry and concentrated to generate 3-alkynes propoxy-phenylcarbinol.

Under 0 ℃, with methylsulfonyl chloride (2.5g, 15.7mmol) and triethylamine (2.8mL, (2.0g 11.0mmol) is dissolved in CH 20mmol) to addcompound 3₂Cl₂In solution in and reaction placed refrigerator, last 16 hours.Processing commonly used provides the mesylate that is light yellow oily.With this oily matter (2.4g, 9.2mmol) be dissolved among the THF (20mL) and add LiBr (2.0g, 23.0mmol).Withreaction mixture heating 1 hour and then it is cooled to room temperature under refluxing.In described mixture, add water (2.5mL) and remove solvent in a vacuum.(3 * 15mL) extracted residues and with saturated NaCl solution (10mL) washing combination organic layer are used Na with ethyl acetate₂SO₄Dry and concentrated to generate the bromide of being wanted.

(1.0g, 0.05mmol Sunbio) are dissolved among the THF (20mL) and with solution and cool off in ice bath with mPEG-OH 20kDa.(6mg, 0.25mmol), simultaneously vigorous stirring number minute period, (2.55g is 11.4mmol) with catalytic amount KI to add the bromide that is obtained by above-mentioned steps then to add NaH.Remove cooling bath and, last 12 hours the extremely backflow of gained mixture heating up.Add water (1.0mL) in the mixture and remove solvent in a vacuum.In resistates, add CH₂Cl₂(25mL) and separate organic layer, with anhydrous Na₂SO₄Drying, and volume is reduced to about 2mL.Dropwise interpolation can cause white depositions in ethereal solution (150mL), collects it to produce the PEG derivative.

Example 17

Poly-(ethylene glycol) polymkeric substance that contains terminal alkynes also can obtain with the reactive molecule coupling that contains alkynes functional group as implied above by making poly-(ethylene glycol) polymkeric substance that contains terminal functional group.N is between 1 and 10.R ' can be the little alkyl of H or C1-C4.

Example 18

(1)

(2)

(2.943g 3.0mmol) is dissolved in CH to make the 4-pentynoic acid₂Cl₂(25mL).(3.80g is 3.3mmol) with DCC (4.66g, 3.0mmol) and the at room temperature described solution of stirred overnight to add N-maloyl imines.Gained crudeproduct NHS ester 7 need not to be further purified and promptly can be used in the following reaction.

Making molecular weight is 5,000Da (mPEG-NH₂, 1g, mPEG-NH Sunbio)₂Be dissolved among the THF (50mL) and and be cooled to 4 ℃ described mixture.By part add a NHS ester 7 (400mg, 0.4mmol), vigorous stirring simultaneously.Mixture was leftstandstill 3 hours, and temperature is to room temperature simultaneously.Then, add water (2mL) and remove solvent in a vacuum.In resistates, add CH₂Cl₂(50mL) and separate organic layer, with anhydrous Na₂SO₄Drying, and volume is reduced to about 2mL.With this CH₂Cl₂Solution is by in part adding ether (150mL).Collect gained throw out and dry in a vacuum.

Example 19

The methylsulfonyl ester of this case representation preparation poly-(ethylene glycol), it also can be called the methanesulfonates (methanesulfonate or mesylate) of poly-(ethylene glycol).Corresponding tosylate and halogenide can be by similar program preparations.

Under nitrogen, with the mPEG-OH in the 150mL toluene (MW=3,400,25g, 10mmol)component distillation 2 hours and solution is cooled to room temperature.With the anhydrous CH of 40mL₂Cl₂And 2.1mL anhydrous triethylamine (15mmol) adds in the solution.Cooling solution and dropwise add 1.2mL in ice bath through distillatory methylsulfonyl chloride (15mmol).Under nitrogen, at room temperature solution stirring is spent the night, and come termination reaction by adding the 2mL dehydrated alcohol.Evaporate described mixture in a vacuum removing solvent (mainly being the solvent except that toluene), filter, concentrate once more in a vacuum and it is deposited in the 100mL ether.With several parts of cold diethyl ether wash filtrates and dry in a vacuum so that mesylate to be provided.

(20g 8mmol) is dissolved among the 75ml THF and with solution and is cooled to 4 ℃ with mesylate.In cooling solution, add sodiumazide (1.56g, 24mmol).Under nitrogen, reaction is heated to backflow lasts 2 hours.Then, evaporating solvent and use CH₂Cl₂(50mL) dilution resistates.With NaCl solution washing organic fraction and use anhydrous MgSO₄Dry.Volume is reduced to 20ml and makes the product precipitation by adding the cold anhydrous ether of 150ml.

Example 20

(1)

(2)

(3)

Use United States Patent (USP) 5,998,595 described methods can produce 4-phenylazide methyl alcohol, described patent is incorporated herein by reference.Under 0 ℃, (2.5g, 15.7mmol) (2.8mL, (1.75g 11.0mmol) is dissolved in CH 20mmol) to add 4-phenylazide methyl alcohol with triethylamine with methylsulfonyl chloride₂Cl₂In solution in and place refrigerator to last 16 hours the reaction.Processing commonly used provides the mesylate that is light yellow oily.This oily matter (9.2mmol) is dissolved among the THF (20mL) and add LiBr (2.0g, 23.0mmol).Reaction mixture is heated to backflow to last 1 hour and then it is cooled to room temperature.In described mixture, add water (2.5mL) and move down and desolventize in vacuum.(3 * 15mL) extracted residues and with saturated NaCl solution (10mL) washing combination organic layer are used anhydrous Na with ethyl acetate₂SO₄Dry and concentrated to generate the bromide of being wanted.

(12mg, (Sunbio) and with bromide (3.32g 15mmol) adds in the mixture together with catalytic amount KI for 2.0g, 0.1mmol 0.5mmol) to handle mPEG-OH 20kDa with the NaH among the THF (35mL).The gained mixture heating up to refluxing, is lasted 12 hours.Desolventize moving down in water (1.0mL) the adding mixture and in vacuum.In resistates, add CH₂Cl₂(25mL) and separate organic layer, with anhydrous Na₂SO₄Drying, and volume is reduced to about 2mL.Dropwise interpolation can cause throw out in ethereal solution (150mL), collects it to produce mPEG-O-CH₂-C₆H₄-N₃

Example 21

With NH₂-PEG-O-CH₂CH₂CO₂(MW 3, and 400Da 2.0g) is dissolved in NaHCO for H₃Be cooled to 0 ℃ in the saturated aqueous solution (10mL) and with solution.Add 3-azido--1-N-maloyl propionic salt (5 equivalent), simultaneously vigorous stirring.After 3 hours, at room temperature add 20mLH₂O and mixture stirred 45 minutes in addition.Use 0.5NH₂SO₄The pH value is adjusted to 3 and add NaCl to about 15 weight % concentration.Use CH₂Cl₂(100mL * 3) extractive reaction mixture is used Na₂SO₄Dry and concentrated.After with the cold diethyl ether precipitation, collect product and dry in a vacuum to generate ω-carboxyl-nitrine PEG derivative by filtering.

Example 22

To as affiliated field in known prepared and in THF, be cooled to and dropwise add mPEG-OMs in-78 ℃ the solution of acetylene lithium (4 equivalent) and be dissolved in solution among the THF, vigorous stirring simultaneously.After 3 hours, make reaction temperature come termination reaction to room temperature and by adding the 1mL butanols.Then, at room temperature add 20mL H₂O and mixture stirred 45 minutes in addition.Use 0.5N H₂SO₄The pH value is adjusted to 3 and add NaCl, up to about 15 weight % concentration.Use CH₂Cl₂(100mL * 3) extractive reaction mixture is used Na₂SO₄Dry and concentrated.After with the cold diethyl ether precipitation, collect product and dry in a vacuum to generate 1-(fourth-3-alkynyloxy group)-methoxy poly (ethylene glycol) (mPEG) by filtering.

Example 23

Use the method described in the following document will contain nitrine-incorporate in the albumen with site selectivity with the amino acid of acetylene: people such as L.Wang, (2001),Science292:498-500; People such as J.W.Chin,Science301:964-7 (2003); People such as J.W.Chin, (2002),Journal of the American Chemical Society124:9026-9027; J.W.Chin ， ﹠amp; P.G.Schultz, (2002),Chem Bio Chem11:1135-1137; People such as J.W.Chin, (2002),PNAS UnitedStates of America99:11020-11024; And L.Wang ， ﹠amp; P.G. Schultz, (2002),Chem.Comm..1-10.In case incorporate amino acid into, under 37 ℃, at 2mM PEG derivative, 1mM CuSO₄And about 1mg Cu line existence lasts 4 hours down with the execution of the 0.01mM albumen in the phosphate buffered saline buffer (PB) (pH 8) cycloaddition reaction.

Example 24

This case description to ethanoyl-D, L-phenylalanine (pAF) and m-PEG-hydroxylamine derivative synthetic.

Use previous Zhang, Z., Smith, B.A.C, Wang, L., Brock, A. be at Cho, C.﹠amp; Schultz, p.G., Biochemistry, the program of describing among (2003) 42, the 6735-6746, synthetic racemize pAF.

For synthetic m-PEG-hydroxylamine derivative, finish follow procedure.To (N-tert-butoxycarbonyl-aminooxy) acetate (0.382g that stirs 1 hour in room temperature (RT) down, 2.0mmol) with 1, (0.16mL, (DCM 70mL) adds methoxyl group-polyoxamide (m-PEG-NH in the solution in to 3-di-isopropyl carbodiimide 1.0mmol) to be dissolved in methylene dichloride₂, 7.5g, 0.25mmol, Mt.30K is available from BioVectra) and diisopropylethylamine (0.1mL, 0.5mmol).To be reflected at RT and stir 48 hours down, and be concentrated into about 100mL then.Dropwise mixture is added in the cold ether (800mL).Be settled out product that tert-butoxycarbonyl is protected and collect, withether 3 * 100mL washing by filtration.Make it be dissolved among the DCM (100mL) again and in ether (800mL) precipitation twice, thereby further it is carried out purifying.Desciccate generates 7.2g (96%) in a vacuum, is confirmed by NMR and Nihydrin test.

Under 0 ℃, in 50%TFA/DCM (40mL), carry out the tert-butoxycarbonyl effect of taking off available from the protected product (7.0g) of above-mentioned steps, last 1 hour and under RT, carried out 1.5 hours then.Remove in a vacuum after most of TFA, the tfa salt of hydroxylamine derivative is converted into HCl salt by the 4N HCl that in resistates, adds in the dioxan (1mL).Make throw out be dissolved among the DCM (50mL) and redeposition in ether (800mL).Collect final product (6.8g, 97%) by filtering, dry in a vacuum withether 3 * 100mL washing, be stored under the nitrogen.Use same program to synthesize other PEG (5K, 20K) hydroxylamine derivative.

Example 25

This case description is used to comprise the hGH polypeptide expression and the purification process of alpha-non-natural amino acid.Host cell is constructed body with quadrature tRNA, quadrature aminoacyl tRNA synthetase and hGH and is transformed.

At first, hanging oneself under 37 ℃, it is a small amount of to take out in the freezing glycerine storing solution that transforms DH10B (fis3) cell, and it is grown in the clear and definite substratum of 2ml composition that contains 100 μ g/ml ampicillins (being supplemented with the glucosyl group basal culture medium of leucine, Isoleucine, trace-metal and VITAMIN).Work as OD₆₀₀When reaching 2-5, be transferred to 60 μ l in the clear and definite substratum of the fresh composition of 60ml that contains 100 μ g/ml ampicillins and under 37 ℃, grow to the OD of 2-5 once more₆₀₀The 50ml culture is transferred in 2 liters of clear and definite substratum of composition that contain 100 μ g/ml ampicillins in 5 liters of fermentor tanks (Sartorius BBI).With salt of wormwood fermentor tank pH value is controlled at pH 6.9, temperature is controlled at 37 ℃, air flow rate is controlled at 5lpm, and with polyalkylene defoamer KFO F119 (Lubrizol) control foam.Reach its maximum value if regulate agitator speed automatically to keep dissolved oxygen content 〉=30% and agitator speed, use pure oxygen to replenish air spray so.At 37 ℃ after following 8 hours, pressing the 50X concentrated solution of speed clear and definite substratum of feed-in composition in culture that index law increases, to keep 0.15 hour-1 particular growth speed.Work as OD₆₀₀Reach at about 100 o'clock, the racemic mixture that adds ethanoyl-phenylalanine is reduced to 28 ℃ to the ultimate density of 3.3mM and with temperature.0.75 after hour, add sec.-propyl-b-D-sulfo-galactopyranoside, up to the ultimate density of 0.25mM.Cell was grown 8 hours down in addition at 28 ℃, and granulation and freezing under-80 ℃ is up to further processing.

The standard His labelled protein purifying procedure that provides by the Invitrogen specification sheets uses ProBondNickel-Chelating Resin (Invitrogen, Carlsbad, CA), come purifying through the mutant hGH of His mark albumen by means of the anionresin tubing string then.

Purified hGH is concentrated into 8mg/ml and is reaction buffer (20mM sodium acetate, 150mM NaCl, 1mM EDTA, pH 4.0) buffer-exchanged.PEG with 20: 1: the hGH molar ratio adds MPEG-oxygen amine powder in the hGH solution.Under 28 ℃, react and last 2 days, simultaneously slight wobble.Come purifying PEG-hGH by unreacted PEG and hGH via the anionresin tubing string.

Before every kind of Pegylation mutant hGH enters experimentation on animals, assess its quality by three calibratings.Under non-reduced condition (Invitrogen), carry out 4-12% acrylamide NuPAGEBis-Tris gel electrophoresis with MES SDS electrophoretic buffer, thereby detect the purity of PEG-hGH.With Ku Masi indigo plant to gel-colored.PEG-hGH bands of a spectrum 95% pure greater than based on photodensitometry scanning.Use is from Charles River Laboratories (Wilmington, KTA MA)²Test kit is examined and determine the endotoxin content of testing among each PEG-hGH by dynamic LAL, and it is less than each dosage 5EU.Assess biological activity and the EC of PEG-hGH with HVI-9 pSTAT5 bioassay (mentioning in the example 2)₅₀Value is less than 15nM.

Example 26

This case description is used to assess the purifying of the hGH polypeptide that comprises alpha-non-natural amino acid and the method for homogeneity.

Fig. 8 is 92 SDS-PAGE that comprise the hGH polypeptide of alpha-non-natural amino acid in the position.The swimming lane 3,4 and 5 of gel is presented at the hGH that position 92 comprises ethanoyl-phenylalanine and is covalently attached to 5kDa, 20kDa or 30kDaPEG molecule.The extra hGH polypeptide that comprises the Pegylation alpha-non-natural amino acid is shown among Figure 11.With each PEG-hGH protein loaded of 5 μ g on each SDS-PAGE.Figure 11 picture A: swimming lane 1, molecular weight marker; Swimming lane 2, WHO rhGH reference standard (2 μ g); Swimming lane 3 and 7,30KPEG-F92pAF; Swimming lane 4,30KPEG-Y35pAF; Swimming lane 5,30KPEG-R134pAF; Swimming lane 6,20KPEG-R134pAF; Swimming lane 8, WHO rhGH reference standard (20 μ g).Figure 11 picture B: swimming lane 9, molecular weight marker; Swimming lane 10, WHO rhGH reference standard (2 μ g); Swimming lane 11,30KPEG-F92pAF; Swimming lane 12,30KPEG-K145pAF; Swimming lane 13,30KPEG-Y143pAF; Swimming lane 14,30KPEG-G131pAF; Swimming lane 15,30KPEG-F92pAF/G120R; Swimming lane 16, WHO rhGH reference standard (20 μ g).Fig. 9 shows the biological activity of the Pegylation hGH polypeptide (5kDa, 20kDa or 30kDa PEG) in the IM-9 molecule; As described in example 2, come manner of execution.

By proteolytic degradation (including, but is not limited to the trypsinase cracking), can assess the purity of hGH-PEG joiner then by mass spectroscopy.PepinskyB. wait the people, J.Pharmcol ﹠amp; Exp.Ther.297 (3): 1059-66 (2001).The method that is used for carrying out tryptic digestion also is described in (2002) the 4th editions the 1938th page of European Pharmacopoeia (European Pharmacopoeia).Described method is revised.The dialysis sample spends the night in 50mM TRIS-HCl (pH 7.5).In 37 ℃ of water-baths, with the trypsinase that 66: 1 mass ratioes are handled through TPCK with trypsin, Worthington) cultivate the rhGH polypeptide, last 4 hours.Culture sample number minute is to stop digestion reaction and to keep 4 ℃ subsequently during HPLC analyzes on ice.To be loaded into 25 * 0.46em Vydac C-8 tubing string (5-μ m particle diameter in 0.1% trifluoroacetic acid through sample digestion (about 200 μ g), 100  apertures) on and under 30 ℃, last 70min with the flow rate of 1ml/min with the gradient of 0-80% acetonitrile and carry out elution.Monitor the elution of tryptic peptide by the absorbancy of 214nm.

The alpha-non-natural amino acid of trypsinase cracking site and arrow indication shown in Figure 10 picture A description has replaces the primary structure (by people such as Becker, the figure that Biotechnol Appl Biochem. (1988) 10 (4): 326-337 revises) of the hGH of F92pAF.Picture B shows the overlapping trypsinase comparison of following each peptide: by peptide (the 30K PEG His of the hGH polypeptide generation that comprises the Pegylation non-naturally encoded amino acids₆-F92pAF rhGH, mark A); Peptide (His by the hGH polypeptide generation that comprises non-naturally encoded amino acids₆-F92pAF rhGH, mark B); And the peptide (WHO rhGH, mark C) that produces by wild-type hGH.WHO rhGH and His₆The relatively demonstration of the trypsinase collection of illustrative plates of-F92pAF rhGH only two peaks move (peptide peak 1 and peptide peak 9) and residual peak identical.Through expressing His₆The N-terminal of-F92pAFrhGH adds His₆Can cause these differences, cause peak 1 to move; And the mobile of peak 9 is by using the phenylalanine to ethanoyl-phenylalanine the position of substitution 92 to produce.Picture C-shows the ratio of enlargement from the peak 9 of picture B.His₆-F92pAF and 30K PEG His₆The relatively demonstration peak 9 of-F92pAF rhGH trypsinase collection of illustrative plates is at His₆Therefore disappear behind-F92pAF rhGH the Pegylation, confirm specific peptide 9 to be modified.

Example 27

This case description is by two homodimers that the hGH polypeptide forms of each self-contained alpha-non-natural amino acid.

Figure 12 comparison 92 comprises His mark hGH polypeptide that ethanoyl-phenylalanine is replaced and apparatus just like the example 25 described IM-9 verification results that are used for the homodimer of the functional group of Pegylation hGH and this modified polypeptide that reactive difunctionality connexon is engaged in the position.

Example 28

This case description serves as the monomer and the dimer hGH polypeptide of hGH antagonist.

Wherein introducing hGH mutain that G120R replaces at site II can be in conjunction with single hGH acceptor, but can not make two receptor dimerizations.Described mutain may be by occupying acceptor site not in the active cells signal transmission path serve as external hGH antagonist (Fuh, people such as G., Science 256:1677-1680 (1992)).Figure 13 picture A shows IM-9 calibrating data, and it measures the pSTAT5 phosphorylation of carrying out with the hGH with G120R replacement.The hGH polypeptide of incorporating alpha-non-natural amino acid at same position (G120) causes also serving as the molecule of hGH antagonist shown in Figure 13 picture B.Construct the dimer of the hGH antagonist shown in Figure 13 picture B, it engages by having the functional group and the reactive difunctionality connexon that are used for Pegylation hGH shown in example 25.Figure 14 shows that this dimer also lacks biological activity in the IM-9 calibrating.

Carry out extra calibrating, its contrast comprises the dimer that G120pAF hGH polypeptide that replaces and the G120pAF that is connected via the PEG connexon modify the hGH polypeptide.WHO hGH inductive STAT5 phosphorylation is by the monomer of dose response scope and the dimer competition that is engaged by the PEG connexon.In addition, carry out surperficial receptor competition research, its displaying monomer and dimer and GH competition are incorporated into the cell surface receptor on IM-9 and rat GHR (the L43R)/BAF3 cell.Dimer serves as than the more effective antagonist of monomer.Table 4 shows the data of these researchs.

Table 4
				Clone	IM-9	IM-9	Rat GHR (IA3R)/BAF3
Calibrating	Suppress PSTAT5	The surface receptor competition	The surface receptor competition
					IC50(nM)	IC50(nM)	IC50(nM)
The G120pAF monomer	3.3	8.4	3.1
				(G120pAF) dimer, the PEG connexon	0.7	2.7	1.4

Example 29

This example has described the active measurement with affinity of the hGH of hGH polypeptide of hGH acceptor in detail.

The clone of rat GH acceptor and purifying the cell foreign lands (GHR ECD, amino acid S29-T238) of rat GH acceptor are cloned between Nde I and Hind III site in the pET20b carrier (Novagen) and with the same framework of C-terminal 6His label.The sudden change of introducing L43 to R is with further near human GH receptor binding site (people such as Souza, Proc NatlAcad Sci USA. (1995) 92 (4): 959-63).By 30 ℃ induced 4-5 hour down with 0.4mM IPTG and in BL21 (DE3) Bacillus coli cells (Novagen) the generation recombinant protein.After dissolved cell, by make the particulate matter resuspending in the Du Ensi utensil that contains 30mL 50mM Tris (pH 7.6), 100mM NaCl, 1mM EDTA, 1%Triton X-100 with described particulate matter washing four times and with the identical buffer reagent washed twice that does not contain Triton X-100.At this moment, inclusion body is made up of the GHR ECD that surpasses 95% and is dissolved in 0.1M Tris (pH 11.5), 2M urea.Aliquots containig by making inclusion body solution by S100 (Sigma) gel-filtration tubing string, reach balance with 50mM Tris (pH 7.8), 1M L-arginine, 3.7mM cystamine, 6.5mM cysteamine and finish again folding.Combination contains the cut of soluble protein and dialyses by 50mM Tris (pH 7.6), 200mM NaCl, 10% glycerine.According to manufacturer specification, make sample of short duration centrifugal to remove any throw out and to cultivate with the aliquots containig of Talon resin (Clontech).With after being supplemented with 20 volume dialysis buffer liquid washing resins of 5mM imidazoles, use the 120mM imidazoles elution albumen in the dialysis buffer liquid.Finally, make the sample dialysed overnight by 50mM Tris (pH 7.6), 30mM NaCl, 1mMEDTA, 10% glycerine, of short duration centrifugal to remove any throw out, be adjusted to 20% glycerine ultimate density, be divided into aliquots containig and be stored under-80 ℃.Use extinction coefficient epsilon=65 of being calculated, 700M^-1* cm^-1, measure protein concentration by OD (280).

The bonded Biocore of GH and GHR^TMAnalyze

The standard amine coupling program of using manufacturers to recommend is fixed in Biacore with the solvable GHR ECD of about 600-800RU^TMOn the CM5 chip.Even this technology can make most of acceptor inactivation, find also that with experimental technique this fixation degree is enough to produce the maximum specific GH association reaction of about 100-150RU, and the binding kinetics that can not notice changes.For example, see people such as Cunningham, JMolBiol. (1993) 234 (3): 554-63 and Wells JA.ProcNatlAcadSci USA (1996) 93 (1): 1-6).

Flow rate injection HBS-EP damping fluid (Biacore with 40 μ l/min^TM, Pharmacia) wild-type of the various concentration in or mutant GH (0.1-300nM) last 4-5 minute and last 15 minutes after injections to monitor and dissociate through the GHR surface.4.5M MgCl by pulse in 15 seconds₂Make surface regeneration.After at least 100 regeneration periods, observe only a small amount of binding affinity loss (1-5%).Use the reference cell that does not contain sessile receptor to subdue any buffering liquid effect and non-specific binding.

With BiaEvaluation 4.1 software (BIACORE^TM) handle kinetics binding data available from the GH titration experiments." divalence analyte " combination model provides gratifying match (usually less than 3 chi²Value), with proposed continuous 1: 2 (GH: GHR) consistent (the Wells JA.Proc Natl Acad Sci USA (1996) 93 (1): 1-6) of dimerization.Ratio (k with indivedual rate constants_Off/ k_On) come calculated equilibrium dissociation constant (Kd).

Table 5 demonstration uses the rat GHR ECD (L43R) that is fixed on the CM5 chip by Biacore^TMThe incorporating parametric that calculates.

Table 5
				GH	kon，×10-5，1/M*s	koff，×104，1/s	Kd，nM
WHOWT	6.4	3.8	0.6
				N-6His?WT	9	5.6	0.6
Rat GH WT	0.33	83	250
				N12pAF	12.5	4.6	0.4
R16pAF	6.8	4.8	0.7
				Y35pAF	7.8	5.3	0.7
E88pAF	6.8	5.4	0.8
				Q91pAF	6.6	4.9	0.7
F92pAJF	8.6	5.0	0.6

R94pAF	5.6	6.0	?1.1
				S95pAF	0.7	3.1	?4.3
N99pAF	2.2	3.8	?1.7
				Y103pAF	～0.06	～6	?＞100
Y111pAF	8.4	4.8	?0.6
				G120R	2.2	22	?10
G120pAF	1.1	23	?20
				G131pAF	6.0	5.3	?0.9
P133pAP	6.4	4.9	?0.8
				R134pAF	8.4	5.8	?0.7
T135pAF	7.2	4.5	?0.6
				G136pAF	6.2	4.3	?0.7
F139pAF	6.8	4.4	?0.7
				K140pAF	7.2	3.7	?0.5
Y143pAF	7.8	6.7	?0.9
				K145pAF	6.4	5.0	?0.8
A155pAF	5.8	4.4	?0.8
				F92pAF-5KD?PEG	6.2	2.3	?0.4
F92pAF-20KD?PEG	1.7	1.8	?1.1
				F92pAF-30KD?PEG	1.3	0.9	?0.7
R134pAF-5KD?PEG	6.8	2.7	?0.4
				R134pAF-30KD?PEG	0.7	1.7	?2.4
Y35pAF-30KD?PEG	0.9	0.7	?0.7
				(G120pAF) dimer	0.4	1.5	?3.4
(F92pAF) dimer	3.6	1.8	?0.5

The GHR stable cell lines

Making IL-3 dependency mouse cell lines BAF3 carry out routine in RPMI 1640, Sodium.alpha.-ketopropionate, penicillin, Streptomycin sulphate, 10% heat-inactivated fetal bovine serum, 50μ M 2 mercapto ethanols and the 10%WEHI-3 clone adjusting substratum as the IL-3 source goes down to posterity.At 5%CO₂Humid atmosphere under, all cells culture is maintained under 37 ℃.

Use BAF3 clone to set up rat GHR (L43R) stabilized cell clone 2E2-2B12-F4.Briefly, with containing the 15 μ g linearizing pcDNA3.1 plasmids of total length rat GHR (L43R) cDNA to 1 * 10⁷The BAF3 cell that individual moderate merges carries out electroporation.The dilution that contains by restriction in the substratum of 800 μ g/ml G418 and 5nM WHO hGH recovered transfectional cell 48 hours before the clone.By antibody (R﹠amp with anti-human GHR; D Systems, Minneapolis MN) carries out that padding comes the transfectant of recognition expression GHR and (BDBiosciences, San Diego analyze on CA) at FACS Array.Then, the proliferation activity of the anti-WHO hGH of transfectant of the good GHR content of screening expression in BrdU propagation calibrating (as mentioned below).In the presence of 1.2mg/ml G418 and 5nMhGH, in the other two-wheeled repetition subclone of want transfectant, set up rat GHR (L43R) cell clone of stable transfection, there is the constant overview of surface receptor expression and multiplication capacity simultaneously.Therefore under the situation that does not have hGH, the cell clone 2E2-2B12-F4 routine of setting up is stored in the BAF3 substratum in addition among the 1.2mg/ml G418.

Breed by the BrdU mark

With 5 * 10⁴The density of individual cells/well is applied to the BAF3 clone 2E2-2B12-F4 of the expression rat GHR (L43R) of serum starvation in the 96 hole culture plates.With the hGH protein activation cell of 12 dose point scopes and use 50 μ MBrdU (Sigma, St.Louis, MO) mark simultaneously.After cultivating 48 hours, atroom temperature use 100 μ l BD cell fixation/Premeabilisation of cells solution (BD Biosciences) to fix/permeation cell, last 30min.For exposing the BrdU epitope, at 37 ℃ of cells of handling fixing/infiltration down with the DNase (Sigma) in 30 μ g/ holes.Carrying out immunofluorescence dyeing with the anti-BrdU antibody (BD Biosciences) that engages with APC can carry out sample analysis on FACS Array.

Table 6 shows as pSTAT5 (IM-9) and BrdU breeds the biological activity of examining and determine the PEG hGH mutant of being summarized.WHO hGH expresses as a whole to contrast between calibrating.

Table 6
			hGH	pSTAT5EC50(nM)	Propagation EC50(nM)
WHOWT	1.0	1.0
			Y35pAF	1.3	1.6±0.8(n＝3)
Y35pAF-30KPEG	10	5.4±2.8(n＝4)
			Y35pAF-40KPEG	53.3	24.0+11.0(n＝3)
F92pAF	2.2±0.4(n＝9)	1.4±0.7(n＝4)
			F92pAF-5KPEG	5.1+0.4(n＝3)	ND
F92pAF-20KPEG	10.5+0.8(n＝3)	ND
			F92pAF-30KPEG	8.8±1.2(n＝8)	4.1±0.9(n＝3)
F92pAF/G120R	＞200,000	＞200,000
			F92pAF/G120R-30KP EG	＞200,000	＞200,000
G131pAF	2.3±1.8(n＝2)	2.1±1.1(n＝3)
			G131pAF-30KPEG	23.8±1.7(n＝2)	4.6±2.4(n＝3)
R134pAF	1.1±0.2(n＝2)	1.7±0.3(n＝3)
			R134pAF-20KPEG	5.3	ND
R134pAF-30KPEG	11.3±1.1(n＝2)	2.5±0.7(n＝4)
			Y143pAF	1.6±0.1(n＝2)	1.8±0.6(n＝2)
Y143pAF-30KPEG	12.3±0.9(n＝2)	6.6±2.7(n＝3)
			K145pAF	2.3±0.5(n＝2)	3.0±1.4(n＝2)
K145pAF-30KPEG	20.6±9.8(n＝2)	5.3±3.5(n＝3)

Example 30

This case description is used to measure the method for the external and activity in vivo of Pegylation hGH.

Cell is in conjunction with calibrating

Under 0 ℃, various concentration (volume: unmarked GH,hGH 10 μ l) or GM-CSF do not exist or exist and¹²⁵Under the situation that I-GH (about 100,000cpm or 1ng) exists, dual multiple culturing cell (3 * 10 in PBS/1%BSA (100 μ l)⁶), (cumulative volume: 120 μ l) that lasts 90 minutes.Then, make cell resuspending and at the ice-cold FCS higher slice of 250 μ l in 350 μ l plastic centrifuge tubes, and centrifugal (1000g; 1 minute).Come the collecting granules thing and in gamma counter (Packard), particulate matter and supernatant liquor are counted respectively by cutting off the pipe end.

Specificity is deducted 100 times of combinations (cpm) (non-specific binding) under the excessive unmarked GH existence in conjunction with the total binding (duplicate mean value) that (cpm) is defined as competing under the non-existent situation of reagent.Measure each the non-specific binding in the used cell type.Use identical¹⁵⁵The I-GH preparation is not experimentizing and described experiment should be showed internal consistency on the same day.¹²⁵I-GH confirms to be bonded to the cell that produces the GH acceptor.Described combination is subjected to the inhibition of unmarked natural GH or hGH in dosage dependence mode, but not suppressed by GM-CSF or other negative control thing.The hGH competition is in conjunction with natural¹²⁵The ability of I-GH is similar to natural GH, illustrates that acceptor discerns two kinds of forms comparably.

Research in the body of Pegylation hGH

PEG-hGH, unmodified hGH and buffered soln are come into operation to mouse or rat.The result shows that Pegylation hGH of the present invention compares the transformation period with excellent activity and prolongation with unmodified hGH, shown in the body weight of remarkable increase.

Measure to engage with do not engage hGH with and the body of varient in the transformation period

In the facility that AAALAC approved and according to the scheme that the Institutional Animal Care and Use Committeeof St.Louis University is ratified, carry out all experimentation on animalies.In having 12 hours bright/dark round-robin rooms, with rat individually stable breeding in cage.Make animal get food qualified Purina rodent solid food 5001 and water arbitrarily.For the rat that hypophysectomizes, tap water additionally contains 5% glucose.

Pharmacokinetic study

Before every kind of Pegylation mutant hGH enters experimentation on animals, assess its quality by three calibratings.Non-reduced condition (Invitrogen, Carlsbad, CA) under, carry out 4-12% acrylamide NuPAGE Bis-Tris gel electrophoresis with MES SDS electrophoretic buffer, thereby detect the purity of PEG-hGH.With Ku Masi indigo plant to gel-colored.PEG-hGH bands of a spectrum 95% pure greater than based on photodensitometry scanning.Use is from Charles River Laboratories (Wilmington, KTA MA)²Test kit is examined and determine the endotoxin content of testing among each PEG-hGH by dynamic LAL, and it is less than each dosage 5EU.Biological activity and the EC50 value of assessing PEG-hGH with IM-9pSTAT5 bioassay (described in the example 2) confirm less than 15nM.

The pharmacokinetic properties of the growth hormone compound that will modify through PEG in available from the male Sprague-Dawley rat (261-425g) of Charles River Laboratories is compared to each other and compares with pegylated growth hormone not.By operation conduit is installed in the carotid artery to be used for blood collecting.Successfully carrying out after conduit installs, before administration with animal TA group (every group of 3-6 only).Through subcutaneous be the 1mg/kg compound of 0.41-0.55ml/kg to the animal dose volume that comes into operation.At each time point, collect in the Eppendorf tube that EDTA applies via inlying catheter collection blood sample and with it.After centrifugal, collect blood plasma and it is stored in-80 ℃, up to analysis.Use available from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, antibody sandwich tethelin ELISA test kit TX) is measured compound concentration.Use comes calculating concentration corresponding to the standard substance of the analogue that is come into operation.The creation facilities program (CFP) that uses a model WinNonlin (Pharsight, version 4.1) estimates pharmacokinetic parameter.Use with the no compartment analysis that linear making progress/the downward trapezoidal integration of logarithm carries out, and concentration data is carried out even weighting.

Figure 15 show to rat come into operation after the single subcutaneous dosage average (+/-S.D.) plasma concentration.Make rat (every group of 3-4 of n=only) take the 1mg/kg hGH wild-type protein (WHO hGH) of single dose, through the hGH of His mark polypeptide (his-hGH) or in the position 92 comprise covalently bound alpha-non-natural amino acid to 30kDa PEG (30KPEG-pAF92 (his) hGH) to ethanoyl-phenylalanine through the hGH of His mark polypeptide.With shown in the timed interval gather plasma sample and examine and determine to analyze described through the injection compound to it.30KPEG-pAF92 (his) hGH has the circulation of comparing significant prolongation with contrast hGH.

Figure 16 show to rat come into operation after the single subcutaneous dosage average (+/-S.D.) plasma concentration.Make rat (every group of 3-6 of n=only) take the 1mg/kg albumen of single dose.Make each position in six different positionss comprise covalently bound alpha-non-natural amino acid to 30kDa PEG to the hGH polypeptide of ethanoyl-phenylalanine and WHO hGH and (his)-hGH relatively.With shown in the timed interval gather plasma sample and examine and determine to analyze described through the injection compound to it.Table 7 shows the pharmacokinetic parameter value that the single dose of hGH polypeptide shown in Figure 16 comes into operation.Shown value is mean value (+/-standard deviation).C_Max: peak concentration; Latter stage t_1/2: terminal half-life; AUC_{0 → inf '}: area under the concentration-time curve is extrapolated to infinitely; Cl/f: apparent total plasma clearance; MRT: mean residence time; Vz/f: the apparent volume that distribute latter stage.

Table 7: the pharmacokinetic parameter value of single dose 1mg/kg subcutaneous administration in the normal male Sprague-Dawley rat

Compound (n)	Parameter
							Cmax (ng/ml)	Latter stage t1/2 (h)	AUC0→inf’ (ngXhr/ml)	MRT(h)	Cl/f (ml/hr/kg)	Vz/f (ml/kg)
	WHO?hGH(3)	529 (+127)	0.53 (+0.07)	759 (+178)	1.29 (+0.05)	1,368 (+327)							1051 (+279)
(his)hGH(4)							680 (±167)	0.61 (±0.05)	1,033 (±92)	1.30 (±0.17)	974 (±84)	853 (±91)
	30KPEG-pAF35(his)hGH(4)	1,885 (±1,011)	4.85 (+0.80)	39,918 (+22,683)	19.16 (±4.00)	35 (+27)							268 (+236)
30KPEG-pAF92(his)hGH(6)							663 (+277)	4.51 (+0.90)	10,539 (+6,639)	15.05 (+2.07)	135 (+90)	959 (+833)
	30KPEG-pAF131(his)hGH(5)	497 (+187)	4.41 (+0.27)	6,978 (+2,573)	14.28 (+0.92)	161 (+61)							1,039 (+449)
30KPEG-pAF134(his)hGH(3)							566 (+204)	4.36 (+0.33)	7,304 (+2,494)	12.15 (+1.03)	151 (+63)	931 (+310)
	30KPEG-pAF143(his)hGH(5)	803 (+149)	6.02 (+1.43)	17,494 (+3,654)	18.83 (+1.59)	59 (+11)							526 (+213)
30KPEG-pAF145(his)hGH(5)							634 (±256)	5.87 (±0.09)	13,162 (±6,726)	17.82 (±0.56)	88 (±29)	743 (±252)

Drug efficacy study

The male Sprague-Dawley rat that hypophysectomizes is available from Charles River Laboratories.Pass through exenterate hypophysis during age in week at 3-4.Make animal adapt to for 3 periods in week, monitor body weight during this period.Be included in that the body weight increment is the animal of 0-8g and it is divided into the treatment group at random in 7 day period before the research.Make rat through subcutaneous take single dose or every day dosage.During studying, every day and continuously rat being weighed, anesthesia, bloodletting and administration (suitably time).Use heparinization kapillary is collected blood and it is positioned in the Eppendorf tube that EDTA applies in the eye socket hole.Take separated plasma and it is stored under-80 ℃, up to analysis by centrifugal.

Figure 17 show to the rat that hypophysectomizes come into operation after the single subcutaneous dosage average (+/-S.D.) plasma concentration.Make rat (every group of 5-7 of n=only) take the 2.1mg/kg albumen of single dose.Be presented at each position in two different positionss and comprise the result of covalently bound alpha-non-natural amino acid to 30kDa PEG the hGH polypeptide of ethanoyl-phenylalanine.With shown in the timed interval gather plasma sample and examine and determine to be used for described through the injection compound to it.

Peptide IGF-1 is the member in somatomedin or the insulin-like growth factor family.Multiple effect in the growth effect of IGF-1 adjusting tethelin.Use the competitive desmoenzyme immunoassays test kit that the rat/mouse IGF-1 standard substance (DiagnosicSystems Laboratories) that is provided is provided to measure IGF-1 concentration.Use two tails distributions, not paired, equal variance, test to determine significant difference by t.Figure 18 picture A shows the assessment of the compound in the rat that hypophysectomizes.Make rat (every group of 5-7 of n=only) through subcutaneous take single dose or every day dosage.Every day, continuously rat is weighed anesthesia, bloodletting and administration (suitably time).Show placebo treatment, wild-type hGH (hGH), 35 and 92 comprise covalently bound body weight result the hGH polypeptide of ethanoyl-phenylalanine to 30kDa PEG through the hGH of His mark ((his) hGH) and in the position.The hGH polypeptide that comprises the Pegylation non-naturally encoded amino acids that Figure 18 picture B is presented at the single dose that comes into operation influences the round-robin of blood plasma IGF-1 content afterwards.Vertical bar is represented standard deviation.In Figure 18 picture A, be different from body weight increment statistically about the 9th day body weight increment of 30KPEG-pAF35 (his) hGH compound, because observe bigger body weight increment about 30KPEG-pAF92 (his) hGH compound.

Figure 18 picture C shows the assessment of the compound in the rat that hypophysectomizes.Make rat (11 every group of n=) through subcutaneous take single dose or every day dosage.Every day, continuously rat is weighed anesthesia, bloodletting and administration (suitably time).Show placebo treatment, wild-type hGH (hGH) and 92,134,145,131 and 143 comprise covalently bound body weight result the hGH polypeptide of ethanoyl-phenylalanine to 30kDa PEG in the position.Figure 18 screen D-graphic demonstration is compared with placebo treatment and wild-type hGH, and the round-robin to blood plasma IGF-1 content after the hGH polypeptide that comprises Pegylation (position 92,134,145,131,143) non-naturally encoded amino acids of the single dose that comes into operation influences.Figure 18 picture E show corresponding to the hGH polypeptide that comprises Pegylation (92,134,145,131,143) non-naturally encoded amino acids average (+/-S.D.) plasma concentration.With shown in the timed interval gather plasma sample and examine and determine to analyze described through the injection compound to it.Vertical bar is represented standard deviation.

Example 31

Comprise the security of Pegylation hGH of non-naturally encoded amino acids and/or the human clinical trial of curative effect

Target is that comparison (includes, but is not limited to Humatrope through subcutaneous Pegylation recombinant human hGH that comprises non-naturally encoded amino acids that comes into operation and commercially available hGH product^TM(Eli Lilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono)) security and pharmacokinetics.

The patient 18 ages is that 20-40 year and body weight are that the healthy volunteer of 60-90kg participates in this research.Described person under inspection will not have the experimental value of significantly unusual clinically hematology or serum chemistry and the screening of negative urine toxicology, HIV screening and hepatitis B surface antigen(HBsAg).It should not have any sign of under conditions: hypertension; Any primary blood disease medical history; Serious liver, kidney, cardiovascular, stomach, urogenital, metabolism, sacred disease medical history; Anaemia or epilepsy medical history; Known bacillary or Mammals are come product-derived, PEG or human serum albumin's allergy; Usually and in a large number drink the human consumer of the beverage that contains caffeine; Participate in any other clinical trial or blood transfusion or donate blood in 30 days of research registration; In 3 months of research registration, be exposed to hGH; Ill in 7 days of research registration; And in 14 days of research registration, before research, there is severely subnormal in physical examination or the clinical experiment assessment.Can assess all person under inspections' security and collect the whole blood gleanings that is used for pharmacokinetic analysis on schedule.All researchs all appoint the approval of Ethics Committee of mechanism and patient's agreement to carry out down.

This research of research and design will be the Phase I of healthy male volunteers, single center, open-label, randomization, two-stage crossing research.18 persons under inspection are appointed as two group (9 person under inspection/groups) in the treatment sequence set at random.Use the Pegylation hGH that comprises non-naturally encoded amino acids and the selected commercially available prod of same dose to carry out quick subcutaneous injection in upper lap, through two independent administration phases GH that comes into operation.The dispensing dosage and the frequency of packaging label indication commercially available prod.Can will use extra administration that the commercially available prod carries out, administration frequency or other to want during the parameter adding studies by comprising extra person under inspection group.Separated each administration phase with 14 days removing phases.For interim each of two administrations, 72 hours (but not between administration phase) was limited to the research centre with the person under inspection after at least 12 hours and the administration before administration.If have extra administration, frequency or other parameter, can add extra person under inspection's group so, equally it is carried out Pegylation hGH test.Approval can be used in this research for the human multiple GH composite that uses.Humatrope^TM(Eli Lilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono)) be the commercially available GH product of approval for human use.The experiment deployment thing of hGH is the Pegylation hGH that comprises non-naturally encoded amino acids.

Blood sampling before the hGH that comes into operation and is afterwards extracted continuous blood by direct venipuncture.In (3 baseline sample) about 30,20 and 10 minutes before the administration and roughly the following time after administration obtains to be used to measure the venous samples can (5mL) of Serum GH concentration: 30 minutes and 1,2,5,8,12,15,18,24,30,36,48,60 and 72 hour.Each serum sample is divided into two aliquots containigs.All serum samples are stored under-20 ℃.On dry ice, consign serum sample.Carry out clinical experiment test (hematology, serum chemistry and urinalysis) on an empty stomach the morning of (at once) and the 19th day before (at once) before the 1st day the predose, the 4th day morning, the 16th day administration.

Bioanalytical method is used to measure Serum GH concentration with ELISA test kit program (Diagnostic Systems Laboratory[DSL], Webster TX).

Security is determined at each administration (the 1st day and the 16th day) 6,24,48 and 72 hour record vital signs at once and after each administration before.Different with baseline during security is measured the incidence be based on adverse events and type and clinical experiment are tested.In addition, also vital signs measurements (comprising blood pressure and physical examination result) different before assessment and the research.

Behind the data analysis self administration of medication in each value each deducts the determined average baselining GH of the mean value concentration of the GH content of three samples of collection when calculating before the administration 30,20 and 10 minutes, thereby proofreaies and correct serum-concentration value after the administration about baseline GH concentration before the administration.If Serum GH concentration is lower than the quantitative levels of calibrating before the administration, so itself and be not included in the calculating of mean value.Determine pharmacokinetic parameter according to serum-concentration data about baseline GH concentration correction.Use the BIOAVL software of latest edition, on Digital Equipment Corporation VAX8600 computer system, calculate pharmacokinetic parameter by the model independent solution.Determine following pharmacokinetic parameter: peak serum concentration (C_Max); Reach the time (t of peak serum concentration_Max); Zero to final blood sampling time (AUC by what use linear trapezoid method then to be calculated from the time_0-72) area under the concentration-time curve (AUC); And by eliminating the transformation period (t the latter stage that computer is calculated according to the elimination factor constant_1/2).By the log-linear concentration-time curve latter stage the consecutive numbers strong point in the linearity region linear regression estimate the elimination factor constant.Calculate mean value, standard deviation (SD) and the variation coefficient (CV) of the pharmacokinetic parameter of each treatment.The ratio of calculating parameter mean value (composite/do not preserve composite) through preserving.

Safety results is equal incidence of distributing adverse events in the treatment group.With clinical experiment test or blood pressure before baseline or the research there is no clinical significantly different, and with research predecessor health check-up come to an end fruit and vital signs measure do not have yet significantly different.The security curve of two treatment groups seems similar.

Pharmacokinetics result makes all 18 persons under inspection (include, but is not limited to Humatrope at the commercially available hGH product that receives single dose at each measured time point^TM(Eli Lilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer) and Saizen/Serostim^TM(Serono)) the average serum GH concentration-time curve (not proofreading and correct about baseline GH content) after one or more product in contrasts with the Pegylation hGH that comprises non-naturally encoded amino acids.All persons under inspection should have baseline GH concentration before the administration in the normal physiological scope.According to determining pharmacokinetic parameter and definite C about the gauged serum data of average baselining GH concentration before the administration_MaxWith t_MaxSelected clinical control reagent (Humatrope^TM(Eli Lilly ﹠amp; Co.), Nutropin^TM(Genentech), Norditropin^TM(Novo-Nordisk), Genotropin^TM(Pfizer), Saizen/Serostim^TM(Serono)) average t_MaxRemarkable t less than the Pegylation hGH that comprises non-naturally encoded amino acids_MaxCompare with the terminal half-life of the Pegylation hGH that comprises non-naturally encoded amino acids, the terminal half-life value of the commercially available hGH product of being tested is significantly less.

Although this research is to carry out in the healthy male person under inspection, expection similarly absorbs feature and security overview and also should be present among other patient group; For example suffer from the patient that sex patient, paediatrics patients with renal failure, the patient in body storage blood scheme or the arrangement of cancer or chronic renal failure are chosen date for operation.

In a word, will be safe and the healthy male person under inspection can well bear through the Pegylation hGH that comprises non-naturally encoded amino acids of the subcutaneous single dose that comes into operation.Based on contrast incidence, clinical experiment value, vital signs and the physical examination result of adverse events, the hGH of commercial form and the security curve that comprises the Pegylation hGH of non-naturally encoded amino acids will be that the Pegylation hGH that comprises non-naturally encoded amino acids of equivalence provides huge clinical efficacy for patient and health care supplier potentially.

Should be appreciated that example as herein described and embodiment just in order to reach the illustrative purpose, and according to its various modifications of making or change and will advise and will be included in the category of the spirit and scope of the application's case and appended claims book the those skilled in the art.The mode that all publications, patent and the patent application case that this paper quotes in order to realize all purposes all quoted in full is incorporated herein.

Table 8: the sequence of being quoted

SEQ ID sequence number	Thesequence title
		1	The full length amino acid sequence ofhGH
2	The mature amino acid sequence of hGH (with merit iso series 1)
		3	Wherein lack the 20-kDa hGH varient of the residue 32-46 ofhGH
21	The nucleotide sequence of total length hGH
		22	The nucleotide sequence of ripe hGH

Sequence table

<110>Cho，Ho?S

Daniel，Thomas

DiMarchi，Richard

Hays，Anna-Maria

Wilson，Troy

Sim，Bee-Cheng

Litzinger，David

＜120〉modified human interferon polypeptides and its purposes

<130>AMBX-0029.00PCT

<150>60/541,528

<151>2004-02-02

<150>60/581,314

<151>2004-06-18

<150>60/581,175

<151>2004-06-18

<150>60/580,885

<151>2004-06-18

<150>60/638,616

<151>2004-12-22

<160>22

<170>PatentIn?version?3.3

<210>1

<211>217

<212>PRT

＜213〉homo sapiens

<400>1

Met?Ala?Thr?Gly?Ser?Arg?Thr?Ser?Leu?Leu?Leu?Ala?Phe?Gly?Leu?Leu

1 5 10 15

Cys?Leu?Pro?Trp?Leu?Gln?Glu?Gly?Ser?Ala?Phe?Pro?Thr?Ile?Pro?Leu

20 25 30

Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg?Ala?His?Arg?Leu?His?Gln

35 40 45

Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu?Glu?Ala?Tyr?Ile?Pro?Lys

50 55 60

Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro?Gln?Thr?Ser?Leu?Cys?Phe

65 70 75 80

Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg?Glu?Glu?Thr?Gln?Gln?Lys

85 90 95

Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu?Leu?Leu?Ile?Gln?Ser?Trp

100 105 110

Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val?Phe?Ala?Asn?Ser?Leu?Val

115 120 125

Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp?Leu?Leu?Lys?Asp?Leu?Glu

130 135 140

Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu?Glu?Asp?Gly?Ser?Pro?Arg

145 150 155 160

Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser?Lys?Phe?Asp?Thr?Asn?Ser

165 170 175

His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr?Gly?Leu?Leu?Tyr?Cys?Phe

180 185 190

Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe?Leu?Arg?Ile?Val?Gln?Cys

195 200 205

Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe

210 215

<210>2

<211>191

<212>PRT

＜213〉homo sapiens

<400>2

Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg

1 5 10 15

Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Glu

20 25 30

Glu?Ala?Tyr?Ile?Pro?Lys?Glu?Gln?Lys?Tyr?Ser?Phe?Leu?Gln?Asn?Pro

35 40 45

Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn?Arg

50 55 60

Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser?Leu

65 70 75 80

Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser?Val

85 90 95

Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr?Asp

100 105 110

Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg?Leu

115 120 125

Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr?Ser

130 135 140

Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn?Tyr

145 150 155 160

Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr?Phe

165 170 175

Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe

180 185 190

<210>3

<211>176

<212>PRT

＜213〉homo sapiens

<400>3

Phe?Pro?Thr?Ile?Pro?Leu?Ser?Arg?Leu?Phe?Asp?Asn?Ala?Met?Leu?Arg

1 5 10 15

Ala?His?Arg?Leu?His?Gln?Leu?Ala?Phe?Asp?Thr?Tyr?Gln?Glu?Phe?Asn

20 25 30

Pro?Gln?Thr?Ser?Leu?Cys?Phe?Ser?Glu?Ser?Ile?Pro?Thr?Pro?Ser?Asn

35 40 45

Arg?Glu?Glu?Thr?Gln?Gln?Lys?Ser?Asn?Leu?Glu?Leu?Leu?Arg?Ile?Ser

50 55 60

Leu?Leu?Leu?Ile?Gln?Ser?Trp?Leu?Glu?Pro?Val?Gln?Phe?Leu?Arg?Ser

65 70 75 80

Val?Phe?Ala?Asn?Ser?Leu?Val?Tyr?Gly?Ala?Ser?Asp?Ser?Asn?Val?Tyr

85 90 95

Asp?Leu?Leu?Lys?Asp?Leu?Glu?Glu?Gly?Ile?Gln?Thr?Leu?Met?Gly?Arg

100 105 110

Leu?Glu?Asp?Gly?Ser?Pro?Arg?Thr?Gly?Gln?Ile?Phe?Lys?Gln?Thr?Tyr

115 120 125

Ser?Lys?Phe?Asp?Thr?Asn?Ser?His?Asn?Asp?Asp?Ala?Leu?Leu?Lys?Asn

130 135 140

Tyr?Gly?Leu?Leu?Tyr?Cys?Phe?Arg?Lys?Asp?Met?Asp?Lys?Val?Glu?Thr

145 150 155 160

Phe?Leu?Arg?Ile?Val?Gln?Cys?Arg?Ser?Val?Glu?Gly?Ser?Cys?Gly?Phe

165 170 175

<210>4

<211>77

<212>DNA

＜213〉Methanococcus jannaschii

<400>4

ccggcggtag?ttcagcaggg?cagaacggcg?gactctaaat ccgcatggcg?ctggttcaaa 60

tccggcccgc?cggacoa 77

<210>5

<211>88

<212>DNA

＜213〉halophilic bacterium kind NRC-1

<400>5

cccagggtag?ccaagctcgg?ccaacggcga?cggactctaa?atccgttctc?gtaggagttc 60

gagggttcga?atcccttccc?tgggacca 88

<210>6

<211>89

<212>DNA

＜213〉halophilic bacterium kind NRC-1

<400>6

gcgagggtag?ccaagctcgg?ccaacggcga?cggacttcct?aatccgttct?cgtaggagtt 60

cgagggttcg?aatccctccc?ctcgcacca 89

<210>7

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>7

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Thr?Tyr?Tyr

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>8

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>8

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Thr?Ser?His

145 l50 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>9

<211>305

<212>PRT

＜213〉Methanococcus jannaschii

<400>9

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Pro?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Ala?Ile?Tyr

145 150 155 160

Leu?Ala?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu?Leu

260 265 270

Glu?Ser?Lsu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>10

<211>305

<212>PRT

＜213〉Methanococcus jannaschii

<400>10

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Pro?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Ile?Pro?Tyr

145 150 155 160

Leu?Pro?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu?Leu

260 265 270

Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>11

<211>305

<212>PRT

＜213〉Methanococcus jannaschii

<400>11

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Lys?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Ala?Ile?Tyr

145 150 155 160

Leu?Ala?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile?His

165 170 175

Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His?Asn

180 185 190

Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser?Lys

195 200 205

Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala?Lys

210 215 220

Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro?Ile

225 230 235 240

Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys?Arg

245 250 255

Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu?Leu

260 265 270

Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys?Asn

275 280 285

Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys?Arg

290 295 300

Leu

305

<210>12

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>12

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Asn?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Pro?Leu?His

145 150 155 160

Tyr?Gln?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>13

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>13

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Lsu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Pro?Leu?His

145 150 155 160

Tyr?Gln?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>14

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>14

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Pro?Val?His

145 150 155 160

Tyr?Gln?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>15

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>15

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Thr

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Ser?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Pro?Ser?His

145 150 155 160

Tyr?Gln?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>16

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>16

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Glu?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Gly?Cys?His

145 150 155 160

Tyr?Arg?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Lel?Glu?Tyr?Pro?Lel?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asu?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>17

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>17

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Leu

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?GIu?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Gly?Thr?His

145 150 155 160

Tyr?Arg?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>18

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>18

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Glu?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Gly?Gly?His

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ile?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>19

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>19

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Ala

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Arg?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Val?Ile?His

145 150 155 160

Tyr?Asp?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>20

<211>306

<212>PRT

＜213〉Methanococcus jannaschii

<400>20

Met?Asp?Glu?Phe?Glu?Met?Ile?Lys?Arg?Asn?Thr?Ser?Glu?Ile?Ile?Ser

1 5 10 15

Glu?Glu?Glu?Leu?Arg?Glu?Val?Leu?Lys?Lys?Asp?Glu?Lys?Ser?Ala?Gly

20 25 30

Ile?Gly?Phe?Glu?Pro?Ser?Gly?Lys?Ile?His?Leu?Gly?His?Tyr?Leu?Gln

35 40 45

Ile?Lys?Lys?Met?Ile?Asp?Leu?Gln?Asn?Ala?Gly?Phe?Asp?Ile?Ile?Ile

50 55 60

Leu?Leu?Ala?Asp?Leu?His?Ala?Tyr?Leu?Asn?Gln?Lys?Gly?Glu?Leu?Asp

65 70 75 80

Glu?Ile?Arg?Lys?Ile?Gly?Asp?Tyr?Asn?Lys?Lys?Val?Phe?Glu?Ala?Met

85 90 95

Gly?Leu?Lys?Ala?Lys?Tyr?Val?Tyr?Gly?Ser?Thr?Phe?Gln?Leu?Asp?Lys

100 105 110

Asp?Tyr?Thr?Leu?Asn?Val?Tyr?Arg?Leu?Ala?Leu?Lys?Thr?Thr?Leu?Lys

115 120 125

Arg?Ala?Arg?Arg?Ser?Met?Glu?Leu?Ile?Ala?Arg?Glu?Asp?Glu?Asn?Pro

130 135 140

Lys?Val?Ala?Glu?Val?Ile?Tyr?Pro?Ile?Met?Gln?Val?Asn?Thr?Tyr?Tyr

145 150 155 160

Tyr?Leu?Gly?Val?Asp?Val?Ala?Val?Gly?Gly?Met?Glu?Gln?Arg?Lys?Ile

165 170 175

His?Met?Leu?Ala?Arg?Glu?Leu?Leu?Pro?Lys?Lys?Val?Val?Cys?Ile?His

180 185 190

Asn?Pro?Val?Leu?Thr?Gly?Leu?Asp?Gly?Glu?Gly?Lys?Met?Ser?Ser?Ser

195 200 205

Lys?Gly?Asn?Phe?Ile?Ala?Val?Asp?Asp?Ser?Pro?Glu?Glu?Ile?Arg?Ala

210 215 220

Lys?Ile?Lys?Lys?Ala?Tyr?Cys?Pro?Ala?Gly?Val?Val?Glu?Gly?Asn?Pro

225 230 235 240

Ile?Met?Glu?Ile?Ala?Lys?Tyr?Phe?Leu?Glu?Tyr?Pro?Leu?Thr?Ile?Lys

245 250 255

Arg?Pro?Glu?Lys?Phe?Gly?Gly?Asp?Leu?Thr?Val?Asn?Ser?Tyr?Glu?Glu

260 265 270

Leu?Glu?Ser?Leu?Phe?Lys?Asn?Lys?Glu?Leu?His?Pro?Met?Asp?Leu?Lys

275 280 285

Asn?Ala?Val?Ala?Glu?Glu?Leu?Ile?Lys?Ile?Leu?Glu?Pro?Ile?Arg?Lys

290 295 300

Arg?Leu

305

<210>21

<211>654

<212>DNA

＜213〉homo sapiens

<400>21

atggctacag?gctcccggac?gtccctgctc?ctggcttttg?gcctgctctg?cctgccctgg 60

cttcaagagg?gcagtgcctt?cccaaccatt?cccttatcca?ggctttttga?caacgctatg 120

ctccgcgccc?atcgtctgca?ccagctggcc?tttgacacct?accaggagtt?tgaagaagcc 180

tatatcccaa?aggaacagaa?gtattcattc?ctgcagaacc?cccagacctc?cctctgtttc 240

tcagagtcta?ttccgacacc?ctccaacagg?gaggaaacac?aacagaaatc?caacctagag 300

ctgctccgca?tctccctgct?gctcatccag?tcgtggctgg?agcccgtgca?gttcctcagg 360

agtgtcttcg?ccaacagcct?ggtgtacggc?gcctctgaca?gcaacgtcta?tgacctccta 420

aaggacctag?aggaaggcat?ccaaacgctg?atggggaggc?tggaagatgg?cagcccccgg 480

actgggcaga?tcttcaagca?gacctacagc?aagttcgaca?caaactcaca?caacgatgac 540

gcactactca?agaactacgg?gctgctctac?tgcttcagga?aggacatgga?caaggtcgag 600

acattcctgc?gcatcgtgca?gtgccgctct?gtggagggca?gctgtggctt?ctag 654

<210>22

<211>576

<212>DNA

＜213〉homo sapiens

<400>22

ttcccaacca?ttcccttatc?caggcttttt?gacaacgcta?tgctccgcgc?ccatcgtctg 60

caccagctgg?cctttgacac?ctaccaggag?tttgaagaag?cctatatccc?aaaggaacag 120

aagtattcat?tcctgcagaa?cccccagacc?tccctctgtt?tctcagagtc?tattccgaca 180

ccctccaaca?gggaggaaac?acaacagaaa?tccaacctag?agctgctccg?catctccctg 240

ctgctcatcc?agtcgtggct?ggagcccgtg?cagttcctca?ggagtgtctt?cgccaacagc 300

ctggtgtacg?gcgcctctga?cagcaacgtc?tatgacctcc?taaaggacct?agaggaaggc 360

atccaaacgc?tgatggggag?gctggaagat?ggcagccccc?ggactgggca?gatcttcaag 420

cagacctaca?gcaagttcga?cacaaactca?cacaacgatg?acgcactact?caagaactac 480

gggctgctct?actgcttcag?gaaggacatg?gacaaggtcg?agacattcct?gcgcatcgtg 540

cagtgccgct?ctgtggaggg?cagctgtggc?ttctag 576

Claims (100)

1. hGH polypeptide that comprises one or more non-naturally encoded amino acids.

2. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprises one or more posttranslational modifications.

3. hGH polypeptide according to claim 1, wherein said polypeptide chain is connected to connexon, polymkeric substance or bioactive molecules.

4. hGH polypeptide according to claim 3, wherein said polypeptide chain is connected to water-soluble polymers.

5. hGH polypeptide according to claim 1, wherein said polypeptide chain are connected to double functional copolymer, difunctionality connexon or at least one extra hGH polypeptide.

6. hGH polypeptide according to claim 5, wherein said difunctionality connexon or polymkeric substance are connected in second polypeptide.

7. hGH polypeptide according to claim 6, wherein said second polypeptide is the hGH polypeptide.

8. hGH polypeptide according to claim 4, wherein said water-soluble polymers comprise poly-(ethylene glycol) part.

9. hGH polypeptide according to claim 4, wherein said water-soluble polymers are connected in the non-naturally encoded amino acids that is present in the described hGH polypeptide.

10. hGH polypeptide according to claim 1, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQID NO:2: 1-5,6-33,34-74,75-96,97-105,106-129,130-153,154-183 and 184-191.

11. hGH polypeptide according to claim 1, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQID NO:2: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (they being described proteic C-terminal) with and any combination.

12. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 29,30,33,34,35,37,39,40,49,57,59,66,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,122,126,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,159,183,186,187 with and any combination.

13. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 29,33,35,37,39,49,57,69,70,71,74,88,91,92,94,95,98,99,101,103,107,108,111,129,130,131,133,134,135,136,137,139,140,141,142,143,145,147,154,155,156,186,187 with and any combination.

14. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 35,88,91,92,94,95,99,101,103,111,131,133,134,135,136,139,140,143,145 and 155 with and any combination.

15. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 30,74,103 with and any combination.

16. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 35,92,143,145 with and any combination.

17. hGH polypeptide according to claim 4, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQID NO:2: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (they being described proteic C-terminal) with and any combination.

18. hGH polypeptide according to claim 17, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 30,35,74,92,103,143,145 with and any combination.

19. hGH polypeptide according to claim 18, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 35,92,143,145 with and any combination.

20. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprises one or more aminoacid replacement, interpolation or disappearance, and it regulates the avidity of described hGH polypeptide to the hGH acceptor.

21. hGH polypeptide according to claim 20, it comprises the aminoacid replacement that is selected from the group that is made up of following each thing: F10A, F10H, F10I among the SEQ ID NO:2; M14W, M14Q, M14G; H18D; H21N; R167N; D171S; E174S; F176Y; I179T; With and any combination.

22. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprises one or more aminoacid replacement, interpolation or disappearance, and it increases the stability or the solubleness of described hGH polypeptide.

23. hGH polypeptide according to claim 22, it comprises the aminoacid replacement G120A among the SEQ ID NO:2.

24. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprises one or more aminoacid replacement, interpolation or disappearance, and it increases in the recombinant host cell or the described hGH polypeptide expression of external synthetic.

25. hGH polypeptide according to claim 24, it comprises the aminoacid replacement G120A among the SEQ ID NO:2.

26. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprises one or more aminoacid replacement, interpolation or disappearance, and it increases the protease resistant of described hGH polypeptide.

27. hGH polypeptide according to claim 26, it comprises the aminoacid replacement that is selected from the group that is made up of the following replacement of C-D intra-annular: R134D, T135P, K140A with and any combination.

28. hGH polypeptide according to claim 1, wherein said non-naturally encoded amino acids to not with described polypeptide in 20 kinds of common amino acids in any one in addition connexon, polymkeric substance or bioactive molecules of reaction have reactivity.

29. hGH polypeptide according to claim 1, wherein said non-naturally encoded amino acids comprise carbonyl, aminooxy, diazanyl, hydrazide group, amino urea groups, azido-or alkynyl.

30. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids comprises carbonyl.

31. hGH polypeptide according to claim 30, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl or be substituted aryl; R₂For H, alkyl, aryl, be substituted alkyl and be substituted aryl; And R₃For H, amino acid, polypeptide or N-terminal are modified base; And R₄For H, amino acid, polypeptide or C-terminal are modified base.

32. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids comprises aminooxy.

33. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids comprises hydrazide group.

34. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids comprises diazanyl.

35. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids residue comprises amino urea groups.

36. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids residue comprises azido-.

37. hGH polypeptide according to claim 36, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl, be substituted aryl or do not exist; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base; And R₃For H, amino acid, polypeptide or C-terminal are modified base.

38. hGH polypeptide according to claim 29, wherein said non-naturally encoded amino acids comprises alkynyl.

39. according to the described hGH polypeptide of claim 38, wherein said non-naturally encoded amino acids has following structure:

Wherein n is 0-10; R₁For alkyl, aryl, be substituted alkyl or be substituted aryl; X is O, N, S or does not exist; M is 0-10; R₂For H, amino acid, polypeptide or N-terminal are modified base; And R₃For H, amino acid, polypeptide or C-terminal are modified base.

40. hGH polypeptide according to claim 4, wherein said water-soluble polymers have the molecular weight between about 0.1kDa and the about 100kDa.

41. according to the described hGH polypeptide of claim 40, wherein said water-soluble polymers has the molecular weight between about 0.1kDa and the about 50kDa.

42. hGH polypeptide according to claim 4, it is by the water-soluble polymers reaction that comprises the amino acid whose hGH polypeptide that contains carbonyl and comprise aminooxy, hydrazine, hydrazides or amino urea groups is made.

43. according to the described hGH polypeptide of claim 42, wherein said aminooxy, hydrazine, hydrazides or amino urea groups are connected in described water-soluble polymers by amido linkage.

44. hGH polypeptide according to claim 4, it is to make by the water-soluble polymers that comprises carbonyl is reacted with the polypeptide that comprises non-naturally encoded amino acids, and described non-naturally encoded amino acids comprises aminooxy, hydrazine, hydrazides or amino urea groups.

45. hGH polypeptide according to claim 4, it is to make by making to comprise the amino acid whose hGH polypeptide that contains alkynes and comprise nitrine water-soluble polymers reaction partly.

46. hGH polypeptide according to claim 4, it is to contain the amino acid whose hGH polypeptide of nitrine and make with the water-soluble polymers reaction that comprises alkynyl moiety by making to comprise.

47. hGH polypeptide according to claim 29, wherein said nitrine or alkynyl are connected in water-soluble polymers by amido linkage.

48. hGH polypeptide according to claim 4, wherein said water-soluble polymers are branch or multiarm polymers.

49. according to the described hGH polypeptide of claim 48, each branch of wherein said branched polymers has the molecular weight between about 1kDa and the about 100kDa.

50. hGH polypeptide according to claim 1, wherein said polypeptide are the hGH antagonist.

51. according to the described hGH polypeptide of claim 50, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: 1,2,3,4,5,8,9,11,12,15,16,18,19,22,25,26,29,65,103,106,107,108,109,112,113,115,116,119,120,123,127,128,129,168,174, before the position 1 (being N-terminal) with and any combination.

52. according to the described hGH polypeptide of claim 50, wherein said polypeptide comprises one or more posttranslational modifications, connexon, polymkeric substance or bioactive molecules.

53. according to the described hGH polypeptide of claim 52, wherein said polymkeric substance comprises the part that is selected from the group that is made up of following each thing: water-soluble polymers; And poly-(ethylene glycol).

54. according to the described hGH polypeptide of claim 50, wherein said non-naturally encoded amino acids is present in the II zone, site of described hGH polypeptide.

55. according to the described hGH polypeptide of claim 50, wherein said polypeptide prevents the dimerization of described hGH acceptor.

56. hGH polypeptide according to claim 1, wherein said non-naturally encoded amino acids comprises sugar moieties.

57. hGH polypeptide according to claim 3, wherein said connexon, polymkeric substance or bioactive molecules are connected in described polypeptide via sugar moieties.

58. comprising at least one, an isolating nucleic acid that is included in the polynucleotide that hybridize to SEQ ID NO:21 or SEQ ID NO:22 under the rigorous condition, wherein said polynucleotide select codon.

59. according to the described isolating nucleic acid of claim 58, wherein said selection codon is selected from the group that is made up of following each thing: amber codon, ocher codon, opal codon, unique password, rare codon and four base codons.

60. a method for preparing hGH polypeptide according to claim 3, described method comprise connexon, polymkeric substance or the bioactive molecules that makes the separation hGH polypeptide contact that comprises non-naturally encoded amino acids comprise the part of reacting with described non-naturally encoded amino acids.

61. according to the described method of claim 60, wherein said polymkeric substance comprises the part that is selected from the group that is made up of following each thing: water-soluble polymers; And poly-(ethylene glycol).

62. according to the described method of claim 60, wherein said non-naturally encoded amino acids comprises carbonyl, aminooxy, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl.

63. according to the described method of claim 60, wherein said non-naturally encoded amino acids comprises carbonyl moiety and described connexon, polymkeric substance or bioactive molecules comprise aminooxy, hydrazine, hydrazides or Urea,amino-part.

64. according to the described method of claim 63, wherein said aminooxy, hydrazine, hydrazides or Urea,amino-part are connected in described connexon, polymkeric substance or bioactive molecules by amido linkage.

65. according to the described method of claim 60, wherein said non-naturally encoded amino acids comprises alkynyl moiety and described connexon, polymkeric substance or bioactive molecules comprise the nitrine part.

66. according to the described method of claim 60, wherein said non-naturally encoded amino acids comprises the nitrine part and described connexon, polymkeric substance or bioactive molecules comprise alkynyl moiety.

67. according to the described method of claim 62, wherein said nitrine or alkynyl moiety are connected in connexon, polymkeric substance or bioactive molecules by amido linkage.

68. according to the described method of claim 61, wherein said poly-(ethylene glycol) part has the molecular-weight average between about 0.1kDa and the about 100kDa.

69. according to the described method of claim 61, wherein said poly-(ethylene glycol) part is branch or multiarm polymers.

70. composition that comprises hGH polypeptide according to claim 1 and pharmaceutically acceptable supporting agent.

71. according to the described composition of claim 70, wherein said non-naturally encoded amino acids is connected in water-soluble polymers.

72. a treatment suffers from the patient's of the illness of regulating by hGH method, its comprise to described patient come into operation the treatment significant quantity according to the described composition of claim 70.

73. cell that comprises according to the described nucleic acid of claim 58.

74. according to the described cell of claim 73, wherein said cell comprises quadrature tRNA synthetic enzyme or quadrature tRNA.

75. method for preparing the hGH polypeptide that comprises non-naturally encoded amino acids, described method comprises: under the condition of the hGH expression of polypeptides that allows to comprise non-naturally encoded amino acids, cultivate the cell that comprises coding hGH polypeptide and comprise one or more polynucleotide, quadrature RNA synthetic enzyme and the quadrature tRNA that select codon; And the described hGH polypeptide of purifying.

76. one kind increases the serum half-life of hGH polypeptide or the method for cycling time, described method comprises with one or more non-naturally encoded amino acids and replaces arbitrary in the described hGH polypeptide or the natural amino acid that exists more than one.

77. one kind by the hGH polypeptide with polynucleotide encoding of sequence shown in SEQ ID NO:21 or the SEQ ID NO:22, wherein said polynucleotide comprise the selection codon, and wherein said polypeptide comprises at least one non-naturally encoded amino acids.

78. according to the described hGH polypeptide of claim 77, wherein said non-naturally encoded amino acids is connected in connexon, polymkeric substance, water-soluble polymers or bioactive molecules.

79. according to the described hGH polypeptide of claim 78, wherein said water-soluble polymers comprises poly-(ethylene glycol) part.

80. according to the described hGH polypeptide of claim 77, wherein said non-naturally encoded amino acids is substituted in the position that is selected from by the group that forms from the following residue of SEQ ID NO:2: before the position 1 (being N-terminal), 1,2,3,4,5,8,9,11,12,15,16,19,22,29,30,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,52,55,57,59,65,66,69,70,71,74,88,91,92,94,95,97,98,99,100,101,102,103,104,105,106,107,108,109,111,112,113,115,116,119,120,122,123,126,127,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,158,159,161,168,172,183,184,185,186,187,188,189,190,191,192 (they being described proteic C-terminal) with and any combination.

81. according to the described hGH polypeptide of claim 77, wherein said non-naturally encoded amino acids comprises carbonyl, aminooxy, hydrazide group, diazanyl, amino urea groups, azido-or alkynyl.

82. according to the described hGH polypeptide of claim 79, wherein said poly-(ethylene glycol) part has the molecular weight between about 0.1kDa and the about 100kDa.

83. according to the described hGH polypeptide of claim 79, wherein said poly-(ethylene glycol) part is branch or multiarm polymers.

84. 3 described hGH polypeptide according to Claim 8, wherein said poly-(ethylene glycol) part has the molecular weight between about 1kDa and the about 100kDa.

85. composition that comprises according to described hGH polypeptide of claim 77 and pharmaceutically acceptable supporting agent.

86. a hGH polypeptide that comprises one or more aminoacid replacement, interpolation or disappearance, described aminoacid replacement, interpolation or disappearance increase the described hGH polypeptide expression in the recombinant host cell.

87. 6 described hGH polypeptide according to Claim 8, it comprises aminoacid replacement G120A.

88. a hGH polypeptide that comprises water-soluble polymers, described polymkeric substance is connected in the single amino acid of described hGH polypeptide by covalent linkage.

89. 8 described hGH polypeptide according to Claim 8, wherein said water-soluble polymers comprises poly-(ethylene glycol) part.

90. 8 described hGH polypeptide according to Claim 8, the wherein said amino acid that is covalently attached to described water-soluble polymers is non-naturally encoded amino acids.

91. according to the described hGH polypeptide of claim 90, wherein said non-naturally encoded amino acids is substituted in the position that is selected from the group that is made up of following position: corresponding to 35,92,143 and 145 of SEQ ID NO:2.

92. hGH polypeptide according to claim 11, wherein said non-naturally encoded amino acids are connected in poly-(ethylene glycol) molecule.

93. according to the described hGH polypeptide of claim 91, wherein said polypeptide further comprises aminoacid replacement G120A.

94. hGH polypeptide that comprises at least one connexon, polymkeric substance or bioactive molecules, wherein said connexon, polymkeric substance or bioactive molecules are connected in described polypeptide by the functional group of non-naturally encoded amino acids, and described non-naturally encoded amino acids is incorporated in the described polypeptide by rrna.

95. according to the described hGH polypeptide of claim 94, wherein said hGH polypeptide is through single Pegylation.

96. a hGH polypeptide that comprises the connexon, polymkeric substance or the bioactive molecules that are connected in one or more non-naturally encoded amino acids, wherein said non-naturally encoded amino acids is incorporated in the described polypeptide by rrna in preselected site.

97. according to the described hGH polypeptide of claim 96, wherein said hGH polypeptide comprises described connexon, polymkeric substance or a bioactive molecules.

98. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprise immunogenic one or more aminoacid replacement, interpolation or the disappearance of regulating described hGH polypeptide.

99. hGH polypeptide according to claim 1, wherein said hGH polypeptide comprise the serum half-life of regulating described hGH polypeptide or one or more aminoacid replacement, interpolation or the disappearance of cycling time.

100. an immunogenic method of regulating the hGH polypeptide, described method comprise with one or more non-naturally encoded amino acids and replace arbitrary in the described hGH polypeptide or the natural amino acid that exists more than one.

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