Drawings
FIG. 1 illustrates a method of delivering a cargo protein via a recombinant protein of the cargo protein and a Protein Transduction Domain (PTD) (Steven R. et al. protein transduction: unrestrained delivery in all Cell trends in Cell Biology, 2000).
FIG. 2 illustrates a method of delivering a cargo protein to the cytosol by endocytosis using a complex of a nanoparticle and the cargo protein (Munish Chanana et al. physiochemical properties of protein-coated gold nanoparticles in biological fluids and cells for and after the same and an advanced cargo diagnostic. Angew. chem. int. Ed.2013).
FIG. 3 illustrates the process of exosome isolation and release from vesicles (MVB) (Graca Raposo and Willemstoorvogel. Exmulticellular vesicles: Exosomes, microviscles, and friends. cell biology 200(4),373-383, 2013).
FIG. 4 illustrates the process of treating cancer by in vivo delivery of siRNA by targeting exosomes (Alvarez-Erviti, L.et al. delivery of siRNA to the mouse blood by system injection obtained exosomes. Nature Biotechnology 29,341-345, 2011).
FIG. 5 illustrates the preparation of a optogenetically designed protein-carrying Exosome (EXPLOR) according to the present invention.
FIG. 6 illustrates the separation of the fusion protein of the cargo protein and the light-specific binding protein inside exosomes when the illumination of EXPLOR is stopped.
FIG. 7 illustrates the intracellular position change of mCherry protein in the transformed HEK293T cells introduced with CIBN-EGFP-CD9 gene and mCherry-CRY2 gene according to blue light irradiation.
FIG. 8 illustrates the experimental procedure for obtaining EXPLOR according to the present invention.
Figure 9 illustrates the results of an assay of the change in the amount of cargo protein (mCherry protein) captured in exosomes according to blue light intensity.
Fig. 10 illustrates the results of a study of the introduction of a cargo protein into a target cell (HT1080) after treatment of the target cell with an exosome comprising the cargo protein (mCherry protein), wherein the left side represents the target cell without treatment with exosome and the right side represents the target cell with exosome treatment.
Fig. 11 is a set of fluorescence images (a) illustrating the results of a study of the introduction of a cargo protein into a target cell (HT1080) after treatment of the target cell with exosomes containing the cargo protein (mCherry protein); and (b) a graph illustrating the results of comparing the rate of apoptotic cells induced by exosome treatment.
FIG. 12 illustrates the intracellular position change of mCherry protein in transformed HEK293T cells introduced with GIGANTEA-EGFP-CD9 gene and mCherry-FKF1LOV upon blue light irradiation.
Figure 13 illustrates the expression of luciferase-mCherry fusion protein measured by fluorescence imaging (a) and luciferase activity and number of molecules in the product cells (b):
comparison: untreated HEK293T cells;
and (3) superposition: HEK293T cells with luciferase-mCherry-CRY 2 introduced only;
XP: HEK293T cells of XPACK-luciferase-mCherry were introduced by using a commercially available vector XPACK (Systems Biosciences) designed for exosome loading technology;
EXPLOR: HEK293T cells into which luciferase-mCherry-CRY 2 and CIBN-EGFP-CD9 according to the present invention were introduced;
FIG. 14 illustrates luciferase activity (a) and number of molecules (b) in the resulting exosomes:
NEG: exosomes produced by untreated HEK293T cells;
and (3) superposition: an exosome produced by HEK293T cells into which luciferase-mCherry-CRY 2 was introduced;
XP: exosomes generated in HEK293T cells introduced into XPACK-luciferase-mCherry by using the commercially available vector XPACK (Systems Biosciences) designed for exosome loading technology;
EXPLOR: an exosome produced by HEK293T cells introduced with the luciferase-mCherry-CRY 2 and CIBN-EGFP-CD9 according to the invention;
the method comprises the following steps: exosomes produced by culturing for 72 hours under 200 μ W blue light irradiation,
no light: exosomes produced by culturing for 72 hours in the absence of light.
FIG. 15 illustrates the loading efficiency of cargo protein in the exosomes generated as described above.
Figure 16 illustrates the transport efficiency of cargo protein into target cells (HeLa) using exosomes:
comparison: exosomes produced by untreated HEK293T cells;
and (3) superposition: an exosome produced by HEK293T cells into which luciferase-mCherry-CRY 2 was introduced;
XP: exosomes produced by introducing HEK293T cells of XPACK-luciferase-mCherry using the commercially available vector XPACK (Systems Biosciences) designed for exosome loading technology;
EXPLOR: introducing an exosome produced by luciferase-mCherry-CRY 2 and HEK293T cells according to the CIBN-EGFP-CD 9;
the method comprises the following steps: exosomes produced by culturing for 72 hours under 200 μ W blue light irradiation,
no light: exosomes produced by culturing for 72 hours in the absence of light.
FIG. 17 illustrates the expression positions of luciferase-mCherry-CRY 2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 18 illustrates the expression positions of Cre-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 19a illustrates the expression of ZsGreen induced by Cre EXPLOR treatment in pCAG-loxP-STOP-loxP-ZsGreen transiently transfected HT1080 cells (scale bar, 40 μm):
negative EXPLOR: exosomes not loaded with cre as negative controls;
cre: EXPLOR: cre-loaded exosomes; and
pCMV-Cre: pCMV-Cre vector transfection was used as a positive control.
FIG. 19b illustrates the expression of ZsGreen induced by treatment with Cre: EXPLOR in pCAG-loxP-STOP-loxP-ZsGreen transiently transfected HeLa cells (scale bar, 40 μm):
negative EXPLOR: exosomes not loaded with cre as negative controls;
cre: EXPLOR: cre-loaded exosomes; and
pCMV-Cre: pCMV-Cre vector transfection was used as a positive control.
FIG. 20 illustrates the expression of ZsGreen induced by treatment with Cre: EXPLOR in pCAG-loxP-STOP-loxP-ZsGreen transiently transfected primary rat embryonic neurons (scale bar, 100 μm):
control EXPLOR: exosomes not loaded with cre as negative controls; and
cre: EXPLOR: cre-loaded exosomes.
FIG. 21 illustrates the induction of ZsGreen expression in transgenic mice harboring the pCAG-loxP-STOP-loxP-eNPR3.0-EYFP gene using Cre: EXPLOR treatment (scale bar, 100 μm):
control EXPLOR: exosomes not loaded with cre as negative controls;
cre: EXPLOR: cre-loaded exosomes;
and Hip: sea horses; and
th: the thalamus.
FIG. 22 illustrates the results of immunohistochemistry for NEuN/GFAP in transgenic mice harboring the pCAG-loxP-STOP-loxP-eNPR3.0-EYFP gene:
pink color: a neuron-specific nucleoprotein; NEuN, positive neuron; and
red: a glial fibrillary acidic protein; GFAP, positive astrocytes.
Objective, 40 ×, scale bar, 20 μm.
FIG. 23 illustrates the expression positions of Cas9-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Figure 24 illustrates the generation of DNA constructs for generating Cas 9-loaded exosomes.
Fig. 25 illustrates the results of determination of the content of the cargo protein (CRISPR-Cas9 protein) captured in exosomes.
FIG. 26 illustrates the expression positions of GBA-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 27 illustrates the expression of endogenous GBA and GBA-mCherry-CRY2 fusion proteins in HEK293T cells, rat primary astrocytes, human primary astrocytes and gaucher fibroblasts transiently transfected with GBA-MCH-CRY2 and CIBN-EGFP-CD 9.
Fig. 28 illustrates the results of determination of the amount of cargo protein (GBA protein) captured in exosomes.
FIG. 29 illustrates the results of an enzyme activity assay for β -glucocerebrosidase, a cargo protein (GBA protein), captured in exosomes:
Exo-Natural: HEK 293T-derived exosomes;
Exo-GBA-exosomes comprising β -glucocerebrosidase.
fig. 30 illustrates the results of treatment of fibroblasts from gaucher's disease with GBA-exosomes, indicating that treatment with GBA-exosomes significantly induced enzymatic activity in β -glucocerebrosidase deficient cells.
Figure 31 illustrates the generation of DNA constructs for the production of PTEN-loaded exosomes and the production of cells stably expressing PTEN-loaded exosomes.
FIG. 32 illustrates the expression positions of luciferase-mCherry-CRY 2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 33 illustrates the results of quantitative determination of luciferase activity based on the number of luciferase molecules.
FIG. 34 illustrates the expression positions of PrxI/II-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Prx I peroxide oxidoreductase I (peroxiredoxin I)
Prx II peroxide oxidoreductase II (peroxiredoxin II)
FIG. 35 illustrates that2O2Protection of PrxI/II loaded exosomes in induced oxidative stress and cytotoxicity.
None: h2O2A group of treatments;
cre: EXPLOR: cre-loaded exosomes;
PrxI: EXPLOR: a PrxI-loaded exosome; and
PrxII: EXPLOR: an exosome loaded with PrxII.
FIG. 36 illustrates the expression positions of MyoD-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 37 illustrates the results of treatment of adipose stem cells with MyoD-loaded exosomes, indicating that cell proliferation was induced 6 days after treatment with MyoD-exosomes (clone # A6).
FIG. 38 illustrates the generation of cells stably expressing p 53-loaded exosomes.
FIG. 39 illustrates the results of the determination of the amount of cargo protein (p53 protein) captured in exosomes.
Stabilizing the cells: cells stably expressing p 53-loaded exosomes;
mcherry: an mcherry-loaded exosome;
p 53: exosomes loaded with p 53.
Fig. 40 illustrates the results of measurement of p53 transcriptional activity using luciferase reporter gene, indicating that treatment with p 53-loaded exosomes induced the transcriptional activity of p53 in doxorubicin-treated HeLa cells.
Figure 41 illustrates the generation of DNA constructs for the production of HMGB 1-loaded exosomes and the production of cells stably expressing HMGB 1-loaded exosomes.
FIG. 42 illustrates the expression positions of srI κ B-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
FIG. 43 illustrates that treatment with srIkB-mCherry: EXPLOR significantly reduced tumor necrosis factor α -induced translocation and DNA binding of NF-. kappa. B p65 subunits in Hela cells.
Figure 44 illustrates the analysis of disease progression following administration of srIkB loaded exosomes to an animal model of rheumatoid arthritis.
Figure 45 illustrates the survival curves of the group treated with srIkB loaded exosomes in the LPS-induced sepsis model.
No exosomes: LPS only treated group;
natural exosomes: group treated with exosomes derived from HEK 293T;
srIkB exosomes: groups treated with srIkB loaded exosomes.
FIG. 46 illustrates the expression positions of pYSTAT3 intracellular antibodies, mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Figure 47 illustrates the delivery of pYSTAT3 intrabodies into cells of a target cell using exosomes loaded with pYSTAT3 intrabodies.
FIG. 48 illustrates the expression positions of Bax-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Figure 49 illustrates that treatment with Bax-loaded exosomes induced rapid release of cytochrome c from mitochondria in Hela cells.
FIG. 50 illustrates the expression positions of AIMP-mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Fig. 51 illustrates the measurement results of the content of cargo protein (AIMP protein) captured in exosomes.
FIG. 52 illustrates the expression positions of mCherry-CRY2 and CIBN-EGFP-CD9 in HEK293T cells, indicating that they share the same position for expression.
Best mode for carrying out the invention
The invention provides a complex comprising exosomes loaded with a cargo protein.
In another embodiment, the invention provides a method of using a light-specific binding protein to make exosomes loaded with a cargo protein.
In a further embodiment, the invention provides a method of delivering a cargo protein to the cytosol using an exosome.
In another embodiment, the invention provides a method for the large scale production of exosomes comprising a fusion protein consisting of an exosome-specific marker and a cargo protein.
The present invention provides methods for the large production of exosomes comprising cargo proteins isolated from exosome membranes by using light-specific binding protein pairs.
The invention also provides a vector for preparing the exosome, and the vector can be used for preparing the exosome.
The invention further provides a method for introducing a cargo protein into the cytosol by using the above exosomes.
In one embodiment, the invention provides pharmaceutical compositions comprising exosomes loaded with cargo proteins and methods of making the same.
in a preferred embodiment, the cargo protein is the super repressor IkB protein that inhibits NF-. kappa.B, Bax (Bcl-2-associated X protein), peroxiredoxin I, peroxiredoxin II, cre recombinase, Cas9 (CRISPR-associated protein 9), Cpf1 (CRISPR 1 from Prevotella (Prevotella) and Francisella (Francisella)), or GBA (β -glucocerebrosidase).
The present invention provides exosomes comprising cargo proteins, which can be used for the treatment of various diseases in vivo by delivering the cargo proteins. For example, exosomes may be prepared to include proteins or sirnas with anti-cancer activity, and then cancer cells treated for cancer therapy (fig. 4).
For exosomes comprising a cargo protein for treating disease, efficient preparation of exosomes is required to have the cargo protein properly loaded. Korean patent publication No.2004-0015508 describes a method for preparing exosomes containing specific antigens. Specifically, it describes a method for releasing a cargo protein by using exosomes, in which a gene encoding a specific antigen is inserted into a host cell line and the protein into which the gene is introduced is stably expressed in the cell line, the protein is released extracellularly by the exosomes, and a method for using the exosomes as a vaccine.
However, exosomes are naturally formed in cells. Therefore, even if a gene encoding a cargo protein is inserted into a cell that endogenously produces exosomes, it is difficult to prepare exosomes comprising the protein expressed therein therefrom.
The present invention provides methods for more efficiently preparing exosomes comprising cargo proteins. As a result, the present inventors succeeded in producing exosomes efficiently comprising a cargo protein by expressing a fusion protein composed of an exosome-specific marker and the cargo protein in large amounts in high-concentration exosome-endogenously producing cells (fig. 5).
According to the above method, the cargo protein is attached to the exosome membrane. Thus, a fusion protein consisting of an exosome-specific marker and cargo protein pair is expressed in high-concentration exosome-producing cells, followed by induction of fusion protein ligation by irradiation. The fusion protein is then introduced into the exosome by the action of an exosome-specific marker. Upon termination of irradiation after introduction, the fusion protein separates into a cargo protein and a light-specific binding protein within the exosome. As a result, exosomes comprising free cargo protein isolated from fusion proteins can be efficiently prepared (fig. 6).
The cargo protein loaded into exosomes of the present invention includes, but is not limited to, native or non-native proteins, truncated forms, or mutated forms. Examples of cargo proteins are listed below, but are not limited thereto.
[ Table 1]
< enzyme >
Enzymes are biocatalytic molecules that accelerate chemical reactions in biological organisms. Enzymes bind to their substrates and can accelerate the reaction rate by reducing their activation. Enzymes can be divided into the following classes: proteases, nucleases, hydrolases, kinases, phosphatases and other types of enzymes.
The target protein loaded in exosome in the present invention includes an enzyme and its regulator. Examples of target proteins are listed in the following description, but are not limited thereto.
-proteases and inhibitors thereof
MMP and TIMP
Matrix Metalloproteinases (MMPs), also known as matrixins (matrixins), are calcium-dependent zinc-containing endopeptidases. MMPs are capable of degrading various extracellular matrix proteins and are known to be involved in the cleavage of cell surface receptors, the release of apoptotic ligands (such as FAS ligand) and chemokine/cytokine inactivation. MMPs are also thought to play a major role in cellular behavior, such as cell proliferation, migration (adhesion/dispersion), differentiation, angiogenesis, apoptosis, and host defense.
Matrix metalloproteinases are inhibited by specific endogenous Tissue Inhibitors of Metalloproteinases (TIMPs), which comprise a family of four protease inhibitors: TIMP1, TIMP2, TIMP3, and TIMP 4.
The balance of MMPs and TIMPs plays an important role in tissue remodeling associated with various physiological or pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis, and metastasis. MMP-2 and MMP-9 are believed to be important in metastasis. MMP-1 is thought to be important in rheumatoid arthritis and osteoarthritis. Dysregulation of the balance between MMPs and TIMPs is also characteristic of acute and chronic cardiovascular diseases.
Exosomes comprising MMPs and TIMPs were prepared by expressing fusion proteins composed of an exosome-specific marker and a target protein in large amounts in high-concentration exosome-producing cells. Exosomes loaded with MMPs or TIMPs are useful for treating MMP-related diseases, including rheumatoid arthritis.
Caspase and inhibitor thereof
caspases (cysteine-aspartic proteases, cysteine aspartase or cysteine-dependent aspartate directed proteases) are a family of proteases that play a major role in programmed cell death, including apoptosis, cell pyroptosis (apoptosis) and necrosis, which forms of cell death are important in protecting organisms from stress signals and pathogenic attack caspases also play a role in inflammation, acting directly on pro-inflammatory cytokines such as pro-IL1 β.
Caspase deficiency has been identified as the cause of tumor development. Tumor growth can occur due to a combination of factors, including mutations in cell cycle genes, which removes the limitation on cell growth; and mutations in apoptotic proteins, such as caspases that respond by inducing cell death in abnormally growing cells.
In contrast, excessive activation of certain caspases, such as caspase-3, can cause excessive apoptosis. This can be seen in several neurodegenerative diseases where nerve cells are lost, such as alzheimer's disease. Caspases involved in processing inflammatory signals are also associated with disease. Insufficient activation of these caspases increases the susceptibility of the organism to infection, as a suitable immune response may not be activated. The important role played by caspases in cell death and disease has led to the study of caspases as drug targets. For example, inflammatory caspase-1 is implicated in causing autoimmune diseases.
Exosomes comprising caspases and their inhibitors are prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high concentration of exosome-producing cells. The exosomes loaded with caspase or its inhibitors may be used to treat caspase-related diseases, including neurodegenerative or autoimmune diseases.
Cathepsin and inhibitors thereof
Cathepsins are proteases found in all animals as well as in other organisms. There are approximately a dozen members of this family, which differ in structure, catalytic mechanism, and the proteins they cleave. Most members are activated at the low pH found in lysosomes. Thus, the activity of this family is almost entirely within these organelles.
Cathepsins are associated with cancer, stroke, alzheimer's disease, arthritis, ebola virus, COPD, chronic periodontitis, pancreatitis and some diseases of the eye including keratoconus. In particular for cancer, cathepsin D is a mitogen that attenuates the anti-tumor immune response of chemotactic factors, thereby inhibiting the function of dendritic cells. Cathepsins B and L are involved in matrix degradation and cell invasion.
Exosomes including cathepsin and its inhibitors are prepared by expressing a fusion protein composed of an exosome-specific marker and a target protein in large amounts in cells producing exosomes at high concentrations. The exosomes loaded with cathepsins or inhibitors thereof are useful for treating various cathepsin-related diseases, including cancer and alzheimer's disease.
Nucleases
Cre recombinase
Cre recombinase is a protein isolated by the P1 phage and induces recombination by detecting two different loxP regions. loxP is a 34bp DNA fragment consisting of two 13bp palindromes at both ends and an 8bp asymmetric nuclear spacer in the middle. The Cre recombinase binds to the palindromic sequence, changes the spacer of the DNA upon cleavage, and then recombines the DNA. The DNA sequence is excised or inverted between two different loxP regions based on the orientation of the spacer. If the loxP regions are in the same or opposite orientation, excision or inversion, respectively, occurs.
[ DNA deletion by Cre recombinase ]
One of representative examples of Cre recombinase utilization is a conditional knockout mouse of tissues that can suppress the mutation phase and expression of a specific gene. This technique removes a specific target gene from some isolated cells by generating a mouse with loxP insertion between the front end and the end of the specific target gene, mating with a transgenic mouse expressing Cre, or directly acting Cre recombinase on specific cells. Conditional knockout mice are capable of efficiently determining the function of a particular gene by expressing that gene, which is lethal during early embryonic development, late embryonic development, or adult life.
The present invention provides exosomes loaded with Cre recombinase protein and demonstrates that Cre recombinase protein is delivered to the cytosol of the target cell. The results indicate that the exosomes of the present invention loaded with the Cre recombinase protein can be used for conditional gene manipulation.
Crispr/Cas9
CRISPR-Cas9 is an artificial RNA-based restriction enzyme that makes DNA correction possible by restricting specific regions of a gene. Recently, it has received much attention as a key element of genetic engineering.
CRISPR is a palindromic sequence, an abbreviated form of clustered regularly interspaced short palindromic repeats (clustered regularly-interspersed short palindromic repeats) and the first observed bacterial acquired immune system. First, the Cas9 protein recognizes and limits invading viruses. The restricted viral sequences are then inserted into the CRISPR sequence and the sequences of the bound virus and CRISPR are transcribed into RNA. This RNA is used to form a Cas9 complex. After this process, the transcribed "CRISPR + viral sequence" binds to Cas9 and removes the same invading virus faster than Cas9 alone. This mechanism can be applied in genetic engineering by restricting the target sequence by binding of the target sequence to the Cas9 complex.
Cpf1 is a protein with similar function to the Cas9 protein in the above engineered endonuclease CRISPR-Cas9 system. As shown below, unlike Cas9, Cpf1 recognizes a Protospacer Adjacent Motif (PAM) sequence. It can be used for regions not recognized by Cas9, in particular it is more practical because the criprpr RNA alone (CrRNA) works. tracrRNA is also required in the case of Cas 9.
[ comparison between Cas9 and Cpf1 proteins ]
The present invention provides exosomes loaded with Cas9 or Cpf1 proteins and demonstrates delivery of Cas9 or Cpf1 proteins to the cytosol of a target cell. The results indicate that the exosomes of the present invention loaded with Cas9 or Cpf1 proteins can be used to remove, add or alter fragments of DNA sequences.
Caspase activated dnase
caspase-activated dnase (CAD) or DNA fragmentation factor subunit β (DFFB) is a protein encoded by the DFFB gene in humans.
Apoptosis is a cell death process that removes toxic and/or unwanted cells during mammalian development, with the process of apoptosis being accompanied by contraction and fragmentation of cells and nuclei and degradation of chromosomal DNA into nucleosome units. DNA Fragmentation Factors (DFFs) are heterodimeric proteins of a 40-kD (DFFB) subunit and a 45-kD (DFFA) subunit. DFFA are substrates of caspase-3 and trigger DNA fragmentation during apoptosis. DFF is activated when DFFA is cleaved by caspase-3. The cleavage fragment of DFFA dissociates from the active component of DFFB. DFFB has been found to trigger DNA fragmentation and chromatin condensation during apoptosis.
An exosome comprising a caspase-activated dnase is prepared by expressing a fusion protein consisting of an exosome-specific marker and a target protein in large amounts in a cell producing the exosome at high concentration. Exosomes loaded with caspase-activated dnase may be used to modulate apoptosis in different systems.
-hydrolases
lysosomal enzymes including β -glucocerebrosidase
one of the common lysosomal storage diseases is Gaucher disease (Gaucher disease), which is induced by a deficiency in the lysosomal enzyme β -Glucocerebrosidase (GBA) gene.
GBA deficiency induces functional disorders in the liver, spleen, bone marrow, etc., by storing glucocerebrosidase/glucosylchingamine on macrophage lysosomes. Hematological abnormalities such as anemia, thrombocytopenia, leukopenia, hepatosplenomegaly (gepatolientalny), osteoclasts, central nerve injury, and the like may also be induced.
The current treatment for gaucher disease is enzyme replacement therapy by intravenous injection of celezyme, a GBA analogue. However, such protein drugs have various disadvantages such as short half-life in blood, low efficiency due to antibody production, difficulty in delivery to lysosomes, and inability to apply neurogenic gaucher disease.
the present invention provides an exosome loaded with GBA (β -glucocerebrosidase) protein and demonstrates that GBA (β -glucocerebrosidase) protein is delivered to the cytosol of a target cell the results indicate that the exosome of the present invention loaded with GBA (β -glucocerebrosidase) protein can be used to treat gaucher disease.
Kinases and phosphatases
Mitogen-activated kinase: p38MAP kinase
P38 mitogen-activated protein kinases are a class of mitogen-activated protein kinases (MAPKs) that respond to stress stimuli, such as cytokines, ultraviolet radiation, heat shock, and osmotic shock. P36MAP kinase is involved in cell differentiation, apoptosis and autophagy.
P38MAP kinase (MAPK) is involved in the signaling cascade that controls cellular responses to cytokines and stress. Potential therapeutic effects of P38 inhibitors on autoimmune diseases and inflammatory processes are being sought.
Exosomes containing p38MAPK and its inhibitors were prepared by expressing large amounts of fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. Exosomes loaded with p38MAPK or inhibitors thereof are useful for treating p38 MAPK-associated diseases, including autoimmune diseases.
Inhibitor kappa B kinase (IKK)
IkB kinase (IKK) is an enzyme complex involved in promoting cellular responses to inflammation.IkB kinase complex is part of the upstream NF-. kappa.B signal transduction cascade.IkB. alpha. (kappa B inhibitor) proteins inactivate NF-. kappa.B transcription factors by masking the Nuclear Localization Signal (NLS) of NF-. kappa.B proteins and keeping them inactive in the cytoplasm.IKK phosphorylates inhibitory IkB. alpha. proteins.
activation of the NF-. kappa.B pathway typically begins in response to various pro-inflammatory stimuli, including lipopolysaccharide expressed on the surface of the pathogen, or release of pro-inflammatory cytokines, such as Tumor Necrosis Factor (TNF) or interleukin-1 (IL-1), following immune cell stimulation, a signaling cascade leads to activation of the IKK complex, an event characterized by binding of NEMO to the cognate kinase subunits IKK- α and IKK- β.
in particular, constitutive NF-. kappa.B activity promotes persistent inflammatory signal transduction at the molecular level and is phenotypically transformed to chronic inflammation.
An exosome comprising IKK is prepared by expressing a fusion protein consisting of an exosome-specific marker and a target protein in large amounts in a cell producing exosomes at high concentrations. The IKK-loaded exosomes are useful for treating NF- κ B-associated diseases, including cancer.
PTEN phosphatase
Phosphatase and tensin homolog (PTEN) are identified as tumor suppressor proteins. Mutation of this gene is a step in the development of many cancers. The protein comprises a tensin-like domain and a catalytic domain similar to a bispecific protein tyrosine phosphatase. Unlike most protein tyrosine phosphatases, this protein preferentially dephosphorylates phosphoinositide substrates. It negatively regulates the intracellular levels of phosphatidylinositol-3, 4, 5-triphosphate in cells and functions as a tumor suppressor by negatively regulating the Akt/PKB signaling pathway.
PTEN deletions or mutations are closely associated with cancer, non-cancerous neoplasia and autism. Especially during tumor development, mutations and deletions of PTEN inactivate it, resulting in increased cell proliferation and decreased cell death. Frequent genetic inactivation of PTEN occurs in glioblastoma, endometrial and prostate cancers; reduced PTEN expression is found in many other tumor types, such as lung and breast cancer. Furthermore, PTEN mutations can also lead to a variety of genetic predisposition to cancer.
Mutations in the PTEN gene have led to several other diseases, such as Cowden syndrome (Cowden syndrome), which is characterized by the occurrence of non-cancerous tumors called hamartomas. These include Bannayan-Lily-Ruvalcaba syndrome (Bannayan-Riley-Ruvalcaba syndrome) and Proteus-like syndrome (Proteus-like syndrome). Diseases caused by PTEN mutations are collectively referred to as PTEN hamartoma syndrome or PHTS. Mutations that cause these syndromes result in the production of proteins that are nonfunctional or non-existent. This defective protein allows cells to divide in an uncontrolled manner and prevents the death of damaged cells, causing tumor growth.
PTEN-containing exosomes are prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. The PTEN loaded exosomes can be used to treat different types of cancer.
Janus kinase
Janus kinases (JAKs) are a family of intracellular non-receptor tyrosine kinases that transduce cytokine-mediated signals through the JAK-STAT pathway. Since members of the type I and type II cytokine receptor families do not possess catalytic kinase activity, they rely on the JAK family of tyrosine kinases to phosphorylate and activate downstream proteins involved in their signal transduction pathways. Upon binding of the receptor to its respective cytokine/ligand, it undergoes a conformational change, bringing the two JAKs close enough to phosphorylate each other.
JAK autophosphorylation induces conformational changes within itself, enabling it to transduce intracellular signals by further phosphorylating and activating transcription factors called STATs (signal transduction and transcription kinases). Activated STATs dissociate from receptors and form dimers before transfer to the nucleus where transcription of selected genes is regulated.
Some examples of molecules that use the JAK/STAT signaling pathway are colony stimulating factors, prolactin, growth hormone, and many cytokines. JAK inhibitors are being developed for the treatment of psoriasis, rheumatoid arthritis, polycythemia vera, alopecia, essential thrombocythemia, ulcerative colitis, myelometaplasia with myelofibrosis and vitiligo.
Exosomes including JAKs and their inhibitors are prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. Exosomes loaded with JAK or inhibitors thereof are useful for treating JAK-associated diseases, including cancer.
-others
Ubiquitin ligases
Ubiquitin ligase (also known as E3 ubiquitin ligase) is a protein that recruits E2 ubiquitin-conjugating enzymes already loaded with ubiquitin, recognizes protein substrates and assists or directly catalyzes the transfer of ubiquitin from E2 to the protein substrates. Ubiquitin is linked to lysine on the target protein by an isopeptide bond. The E3 ligase interacts with the target protein and the E2 enzyme, thus conferring substrate specificity for E2.
Ubiquitination of E3 ligase has profound significance in cell biology in a number of areas such as cell trafficking, DNA repair, and signal transduction. E3 ligase is also a key player in cell cycle control, mediating the degradation of cyclins as well as cyclin-dependent kinase inhibitors.
E3 ubiquitin ligase regulates homeostatic balance, cell cycle and DNA repair pathways, and thus, many of these proteins are involved in a variety of cancers, including the well-known MDM2, BRCA1 and Von Hippel-Lindau tumor inhibitors. For example, mutations in MDM2 have been found in gastric, renal cell and liver cancers (among others), resulting in increased transcription of MDM2mRNA by increasing the affinity of the MDM2 promoter for the Sp1 transcription factor, thereby deregulating MDM2 concentrations.
Exosomes containing ubiquitin ligase are prepared by expressing in large quantities fusion proteins consisting of an exosome-specific tag and a target protein in high concentration exosome-producing cells. The exosomes carrying ubiquitin ligase can be used for treating ubiquitination-related diseases, including cancer.
Luciferase enzyme
Luciferases are a generic term for a class of oxidases that produce bioluminescence, and are generally distinct from photoproteins. Luciferases are widely used in biotechnology, microscopy and as reporter genes, and many of the same applications as fluorescent proteins. However, unlike fluorescent proteins, luciferases do not require an external light source, but do require the addition of luciferin (a consumable substrate).
All luciferases are classified as oxidoreductases (EC 1.13.12.-), which means that they act on a single donor by binding molecular oxygen. Since luciferases come from many unrelated distinct protein families, there is no unified mechanism, as any mechanism relies on the combination of luciferase and luciferin. However, all characterized luciferase-luciferin reactions to date have shown the need for molecular oxygen at some stage.
In biological studies, luciferases are commonly used as reporters to assess transcriptional activity in cells transfected with genetic constructs containing the luciferase gene under the control of a promoter of interest. In addition, pro-fluorescent molecules (pro-fluorescent molecules) that convert to luciferin at the activity of a particular enzyme can be used to detect enzyme activity in coupled or two-step luciferase assays. Such substrates have been used to detect caspase activity, cytochrome P450 activity, and the like.
The luciferase may also be used to detect cellular ATP levels in a cell viability assay or a kinase activity assay. Luciferase can be used as an ATP sensor protein by biotinylation. Biotinylation luciferase was immobilized on the cell surface by binding to a streptavidin-biotin complex. This allows the luciferase to detect the efflux of ATP in the cells and will effectively show the real-time release of ATP by bioluminescence. Luciferase may additionally be more sensitive to the detection of ATP by altering certain amino acid residues in the protein sequence to increase the luminescence intensity.
Whole animal imaging (referred to as in vivo or occasionally ex vivo imaging) can be performed using injection of luciferase-expressing cell lines. Different types of cells (e.g., bone marrow stem cells, T cells) can be designed to express luciferase, allowing it to be non-invasively visualized in a living animal using a sensitive charge coupled device camera (CCD camera). This technique has been used to track tumorigenesis and tumor response to treatment in animal models.
The present invention prepared exosomes loaded with luciferase protein and confirmed that luciferase protein was delivered to the cytosol of the target cell. The results indicate that the exosomes of the present invention loaded with luciferase protein can be used for cell viability assays, kinase activity assays and whole animal imaging.
Peroxide oxidoreductases
The peroxiredoxin (Prx) is a typical antioxidant enzyme in cytoplasm, and 0.1-0.8% of water-soluble protein is obtained in mammalian cells. Prx has the ability to reduce hydroperoxides to H by accepting 2 e-in cells2The role of O and ROH-. By participating in H2O2(nmol concentration) formation and removal, Prx is also involved in cell proliferation, differentiation, death and cell signaling. Based on the number of cysteine amino acids, Prx is more specifically classified as 1-Cys Prx or 2-Cys Prx. Furthermore, 2-Cys prx is subdivided into "classical" or "atypical" based on differences in structure and mechanism. All three Prx differ in redox in the secondary process of Cys-SOH formation. Prx I-Prx IV are typically 2-Cys Prx, Prx V is atypical 2-Cys Prc, and Prx VI is 1-Cys Prx. Certain 2-Cys Prx form oligomers.
H production by modulating growth factor and TNF- α in cells2O2And Prx I and II are involved in activation of receptor signaling pathways. In particular, Prx II has the effect of protecting cells from stimulation by cell death-inducing factors such as serum starvation, ceramide and etoposide (epotoside).
In normal cells, Prx I maintains PTEN phosphatase activity by inhibiting oxidation of PTEN phosphatase. However, in the case of increased oxidative stress, H2O2By irreversible oxygenMethylation isolates Prx from PTEN, thereby inhibiting PTEN activity. Thus, it induces tumors by persistently activating cell proliferation signals such as Akt.
Changes in the amount of Prx in a cell have a significant relationship with disease. The change of the active oxygen amount has close connection in the processes of cancer, arteriosclerosis, respiratory inflammation, osteoporosis, obesity, degenerative dementia development and the like.
The present invention provides exosomes loaded with peroxide oxidoreductase I or peroxide oxidoreductase II proteins and demonstrates that the peroxide oxidoreductase I or peroxide oxidoreductase II proteins are delivered to the cytosol of a target cell. The results indicate that the exosomes of the present invention loaded with either a peroxiredoxin I or a peroxiredoxin II protein can be used for treating reactive oxygen species related diseases.
< transcription factor >
Transcription factors are proteins that regulate mRNA transcribed from DNA in eukaryotes. Transcription factors are involved in basic transcriptional regulation, organism development, responses to intercellular signals or environments, cell cycle control, and pathogenesis.
The target proteins loaded in the exosomes of the present invention include transcription factors and their regulators (enhancers or repressors). Examples of target proteins are listed in the following description, but are not limited thereto.
Transcription factors and their regulators
NF-kB regulator, super repressor IkB
NF- κ B is a major transcription factor inducing inflammatory response and regulates the expression of inflammation-associated genes in various cells, particularly immune cells. Thus, selective inhibition of the NF- κ B signaling pathway that is overactive in immune cells may be an effective therapeutic strategy for incurable chronic inflammatory diseases, such as rheumatoid arthritis, sepsis and psoriasis. In addition, activation of NF-. kappa.B has the effect of inhibiting apoptosis by increasing the expression of anti-apoptotic factors. From this effect, sustained activation of the NF- κ B signaling pathway in cancer is responsible for resistance to anticancer drugs and subsequently reduces the therapeutic effect of anticancer drugs.
the majority of NF-. kappa.B is in an inactive state by binding to the NF-. kappa.B suppressor protein IkB in normal cells.IkB kinase (IKK) complexes activated by various stimuli such as TNF- α and LPS phosphorylate I.kappa.B is then phosphorylated, phosphorylated I.kappa.B is ubiquitinated and eventually degraded by proteasomes.NF-. kappa.B (p50/p65) bound to I.kappa.B crosses the nuclear membrane by degradation of I.NF-. kappa.B (p50/p65) activates mRNA transcription by binding to the promoter region of nuclear target genes after passing, which are important elements of the immune response that induce transcription of cytokines and inflammatory mediators such as iNOS, COX-2, NO, PGE2, TNF- α and IL-1 (Lappas et al, biol. reprod.67: 668673, 2002).
The superseprder I κ B is a S32A and S36A mutant form of I κ B that may persistently inhibit NF- κ B because it is not phosphorylated by I κ B kinase and is not degraded by proteasomes. Therefore, its potential as a treatment for various inflammatory diseases is great.
The present invention provides exosomes loaded with the supersuppressor ikb protein and demonstrates that the supersuppressor ikb protein is delivered to the cytosol of a target cell. The results indicate that the exosomes of the present invention loaded with the super repressor ikb protein can be used for the treatment of inflammatory diseases.
MyoD
MyoD is a protein that plays a key role in regulating muscle differentiation. MyoD belongs to a family of proteins called Myogenic Regulators (MRF). MyoD is known to have binding interactions with hundreds of muscle gene promoters and to allow myoblast proliferation. One of the major functions of MyoD is to remove cells from the cell cycle by enhancing transcription of p21 and myogenin.
Exosomes comprising MyoD proteins are prepared by expressing in large quantities a fusion protein consisting of an exosome-specific marker and a target protein in cells producing exosomes at high concentrations. The MyoD-loaded exosomes may be used to treat myoblast-related diseases.
Tbx18(T-box transcription factor 18)
Tbx18 encodes a member of the evolutionarily conserved family of transcription factors, and plays a crucial role in embryonic development. Tbx18 is characterized by the presence of a DNA-binding T-box domain and belongs to the vertebrate specific Tbx1 subfamily. Tbx18 acts as a transcriptional repressor by antagonizing transcriptional activators in the T-box family. Tbx18 is required during various developmental processes in tissues and organs, including the heart and coronary vessels, ureters, and spine. Tbx18 is also required in the sinoatrial node (SAN) header area.
Tbx18 transduction is a method of opening genes in cardiomyocytes as a means of treating certain arrhythmias. In a healthy heart, the sinoatrial node cells act as pacemakers of the heart and cause the heart to beat regularly. A problem with sick sinus syndrome is that the SA node does not function properly, resulting in arrhythmia. Expression of Tbx18 into atrial myocytes using adenovirus converted atrial myocytes into SA node cells that initiate heartbeats. Tbx18 may be one of a number of forms of gene therapy for treating cardiac arrhythmias.
Exosomes comprising Tbx18 protein were prepared by expressing fusion proteins composed of an exosome-specific marker and a target protein in large quantities in high-concentration exosome-producing cells. Exosomes loaded with Tbx18 protein are useful for treating sick sinus syndrome.
P53
The tumor protein p53 is known to be the guardian of the genome, as it preserves the stability of the genome by preventing genomic mutations. When DNA is damaged, p53 can activate DNA repair proteins. In addition, p53 can inhibit growth by keeping the cell cycle at the G1/S regulatory point of DNA damage recognition. In the case of DNA damage and irreparable, p53 can induce apoptosis. Finally, p53 is critical for the senescence response of short telomeres. p53 is activated in response to a myriad of stresses including DNA damage, oxidative stress, osmotic shock, ribonucleotide consumption and deregulated oncogene expression.
If p53 is impaired, tumor suppression is severely compromised. A person who inherits only one functional copy of the P53 gene is likely to develop a tumor in early adulthood. Increasing the amount of p53 may be a solution to treat tumors or prevent their spread.
Exosomes comprising the p53 protein were prepared by expressing in large quantities a fusion protein consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. The exosome loaded with the p53 protein can be used to treat various types of cancer.
HMGB1 (high mobility group protein B1)
Like histones, HMGB1 is one of the most important chromatin proteins. In the nucleus, HMGB1 interacts with nucleosomes, transcription factors, and histones. This nucleoprotein organizes DNA and regulates transcription. After binding, HMGB1 bends the DNA, which favors the binding of other proteins. It also interacts with nucleosomes to loosen stacked DNA and remodel chromatin.
HMGB1 is secreted by immune cells through the amitoses pathway. Activated macrophages and monocytes secrete HMGB1 as a cytokine mediator of inflammation. Antibodies that neutralize HMGB1 confer protection against damage and tissue injury during arthritis, colitis, ischemia, sepsis, etc.
Exosomes comprising HMGB1 protein were prepared by expressing fusion proteins composed of an exosome-specific marker and a target protein in large amounts in high-concentration exosome-producing cells. The exosome carrying the HMGB1 protein can be used for treating inflammatory diseases.
NeuroD1
neurogenic differentiation 1 (also known as β 2) is a transcription factor of the NeuroD type, which mediates transcriptional activation by binding to the promoter consensus core sequence 5'-CANNTG-3' containing E box, which helps to regulate several cellular differentiation pathways, which promote early retinal ganglion cells, inner ear sensory neurons, granulosa cells forming the dentate gyrus layer of the cerebellum or hippocampus, endocrine islet cells of the pancreas, and enteroendocrine cells of the small intestine.
Exosomes comprising NeuroD1 protein were prepared by expressing fusion proteins composed of an exosome-specific marker and a target protein in large quantities in cells producing exosomes at high concentrations. Exosomes loaded with NeuroD1 protein can be used to modulate neuronal development.
Tumor-associated macrophages (TAMs) are cells belonging to the macrophage lineage. They are located near or within the tumor mass. TAMs are derived from circulating monocytes or fixed tissue macrophages, which form the major leukocyte infiltrate found within the stroma of many tumor types. TAMs are associated with poor prognosis in breast, ovarian, glioma and lymphoma types, with better prognosis in colon and stomach cancers, and with both good and poor prognosis in lung and prostate cancer.
TAMs are divided into two major phenotypes, M1 and M2. M1 TAMs inhibit cancer progression, while M2TAMs promote cancer progression. Several transcription factors are associated with the conversion of M2 macrophages to M1 macrophages. The target protein loaded in the exosome of the present invention includes transcription factors involved in the M2 to M1 conversion of macrophages. Examples of target proteins are listed in the following description, but are not limited thereto.
IRF5
IRF5 is a member of the interferon regulatory factor (a class of transcription factors). It plays an important role in the activation of virus-mediated interferons and the regulation of cell growth, differentiation, apoptosis and immune system activity. IRF5 works by interacting directly with DNA or other proteins.
IRF5 acts as a molecular switch that controls whether macrophages promote or inhibit inflammation. Blocking IRF production in macrophages may help treat a wide range of autoimmune diseases, and up-regulating IRF5 levels may help treat people with weak or impaired immune systems.
Exosomes comprising IRF5 protein were prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. The exosome loaded with IRF5 protein can be used for macrophage transformation from M2 to M1 for the treatment of various types of cancer.
IRF3
IRF3 is a member of the interferon regulatory factors and is a group of transcription factors. IRF3 includes a functional domain, a nuclear export signal, a DNA binding domain, a C-terminal IRF-associated domain, and several regulatory sites. It is present in an inactive form in the cytoplasm of uninfected cells. Upon viral infection, double stranded RNA or Toll-like receptor signaling, IKBKE and TBK 1 kinases phosphorylate them. This leads to dimerization and nuclear localization. IRF3 can activate different gene expression programs in macrophages.
Exosomes comprising IRF3 protein were prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. The exosome loaded with IRF3 protein can be used for macrophage transformation from M2 to M1 for the treatment of various types of cancer.
STAT1
STAT1 can be activated by several ligands such as interferon α, interferon gamma, epidermal growth factor, platelet-derived growth factor, or interleukin 6.
Upon binding of type I IFNs to cell surface receptors, Jak kinases are activated and phosphorylate STAT1 and STAT 2. STATs dimerize and bind to ISGF3G/IRF-9 to form a complex called ISGF3 transcription factor. ISGF3 binds to IFN-stimulated response elements to activate transcription of IFN-stimulated genes.
STAT1 is a tyrosine and serine phosphate in response to type II IFNs. It forms a homodimer and binds to IFN γ -activated sequences to drive expression of target genes, inducing a cellular antiviral state.
Exosomes comprising STAT1 protein were prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. Exosomes loaded with STAT1 protein may be used to treat various types of cancer.
SOCS3
Inhibitors of cytokine signaling are members of STAT-induced STAT inhibitors. STAT-induced STAT inhibitors are cytokine-induced negative regulators of cytokine signaling. SOCS3 is induced by various cytokines such as IL6, IL10 and IFN- γ.
Overexpression of SOCS3 inhibited insulin signaling in adipose tissue and liver, but not in muscle. However, the absence of SOCS3 in the skeletal muscle of mice prevented obesity. SOCS3 also contributes to leptin resistance and insulin resistance due to increased ceramide synthesis. Studies have shown that removal of the SOCS gene can prevent insulin resistance in obesity. SOCS3 protein can bind to JAK2 kinase and inhibit the activity of JAK2 kinase.
Exosomes comprising SOCS3 protein were prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in high-concentration exosome-producing cells. The exosome loaded with SOCS3 protein can be used to treat various types of cancer.
< antibody >
Antibodies are proteins that recognize and bind to their specific antigen through the "Y" shaped antibody tip Fab variable region. The antibody can inhibit the activity of the target antigen protein by binding thereto.
Target proteins loaded in the exosomes of the present invention include antibodies and antibody-related peptides. Examples of target proteins are listed in the following description, but are not limited thereto.
Antibodies and related peptides
pySTAT3 intrabodies
STAT (signal transduction and transcription) is a well-established transcription factor: STAT1, STAT2, STAT3, STAT4, STAT5(STAT5A and STAT5B), and STAT 6. STAT3 protein has a C-terminal transcriptional activation domain (the major phosphorylation sites of STAT3 are tyrosine 705 and serine 727 residues). Tyrosine phosphorylation and subsequent STAT3 dimerization facilitate transport to the nucleus and activation of transcription.
The JAK/STAT3 signaling pathway is recognized in the activation of growth factor-induced interferon signaling and is involved in proliferation, differentiation, apoptosis, angiogenesis, tumorigenesis and immunity. Therefore, the STAT3 protein can be used as a good target point of a single drug or a combined therapeutic drug for developing an anti-cancer drug.
The present invention prepared exosomes carrying the ystat3 intrabodies and confirmed that the ystat3 intrabodies were delivered to the cytosol of the target cells. The results indicate that exosomes of the present invention loaded with the cystat 3 intrabodies can be used to treat cancer.
< Others >
Apoptosis-related proteins
Apoptosis (programmed cell death) is a process by which damaged cells are removed by various factors, and abnormal apoptosis induces tumorigenesis. For this reason, studies on the induction of apoptosis of tumor cells have been actively progressed as a strategy for removing tumors. Chromatin condensation, apoptotic body formation and DNA fragmentation caused by cell atrophy are characteristic of apoptosis. Apoptosis is induced by two different pathways; one is the intrinsic pathway through the mitochondria and the other is the extrinsic pathway through the death receptor. Apoptosis is regulated by various factors, such as activation of the pro-apoptotic Bcl-2 family, cleavage of procaspases, and fragmentation of poly-ADP-ribose polymerase (PARP), among others. In particular caspases belonging to cysteine proteases are pro-enzymes in normally proliferating cells and are activated by apoptosis-inducing signals and then have a significant role in apoptosis by the inclusion of cargo proteins such as PARP.
Most apoptotic stimuli induce apoptosis in mammalian cells via pathways controlled by members of the Bcl-2 gene family, which encodes a homologous group of proteins including apoptotic agonists and antagonists, such as Bcl-2 and Bcl-XL. Even if these members are differentially regulated, the members share a domain of sequence homology. During apoptosis, the anti-apoptotic or pro-apoptotic effects of Bcl-2 and Bax (21% identity to Bcl-2 at the protein level) are regulated by homo-and heterodimers, with the difference being the ratio of Bcl-2 to Bax.
Pro-apoptotic proteins: bax
Bax (Bcl-2 related X protein) is one of the Bcl-2 protein family, the so-called Bcl-2-like protein 4. Bax binds to the outer mitochondrial membrane, and 4 residues at the C-terminus protrude into the mitochondrial membrane space, and Bax has the effect of activating apoptosis. Specific information on the above proteins and their gene base sequences is published on NCBI (GenBank: NM-001291428, NP-001278357, etc.).
Bax is one of the Bcl-2 gene families that synthesizes pro-apoptotic proteins. Bax represses its transcription by mutant p 53. It is known that the insertion or deletion of a base sequence of Bax is responsible for a significant decrease in Bax expression in cell lines of hematological, colon and rectal cancers.
Bax is known to be involved in apoptosis of neuronal development, homeostatic balance of lymphatic and reproductive systems, cell death due to DNA damage, ischemia reperfusion injury, and the like.
The present invention provides exosomes loaded with Bax (Bcl-2 related X protein) protein and demonstrates delivery of Bax (Bcl-2 related X protein) protein to the cytosol of a target cell. The results indicate that the exosomes of the present invention loaded with Bax (Bcl-2 related protein X) protein can be used for treating cancer.
Anti-apoptotic proteins: Bcl-xL
B-cell lymphoma encoded by BCL 2-like 1 gene-extra large (BCL-xL) is a transmembrane molecule in mitochondria. Bcl-xL is a member of the Bcl-2 protein family and acts as an anti-apoptotic protein by preventing the release of mitochondrial contents, such as cytochrome c, which causes the activation of caspases and ultimately leads to programmed cell death.
One accepted concept in the field of apoptosis is that the relative amounts of pro-survival and anti-survival Bcl-2 family proteins determine whether a cell will undergo cell death; if more Bcl-xL is present, the cell will survive if the well is impermeable to pro-apoptotic molecules. Like Bcl-2, Bcl-xL is involved in the survival of cancer cells by inhibiting the function of tumor suppressor p 53.
Exosomes comprising Bcl-xL proteins are prepared by expressing in large quantities fusion proteins consisting of an exosome-specific marker and a target protein in cells producing exosomes at high concentrations. The exosomes loaded with Bcl-xL protein can be used to modulate apoptosis.
-others
Multifunctional signal molecules: AIMP (multifunctional protein interacting with aminoacyl tRNA synthetase)
multifunctional protein 1 of the amide-tRNA synthetase interaction (AIMP1) is a non-catalytic component of the multi-synthase complex, stimulates the catalytic activity of cytoplasmic arginyl-tRNA synthase, has inflammatory cytokine activity, stabilizes SMURF2 by binding to SMURF2 and inhibiting its SMAD 7-mediated degradation, negatively regulates TGF- β signaling, participates in the homeostatic balance of glucose by inducing glucagon secretion at low glucose levels, promotes dermal fibroblast proliferation and wound repair.
Plays a role in angiogenesis by inducing endothelial cell migration at low concentrations and apoptosis at high concentrations. Inducing dendritic cell maturation and monocyte adhesion. Degradation of HIF-1A by interaction with PSMA7 modulates endothelial cell responses.
Multifunctional protein 2(AIMP2) interacting with aminoacyl tRNA synthetases is essential for the assembly and stability of aminoacyl tRNA synthetase complexes. Mediates ubiquitination and degradation of FUBP 1, a transcriptional activator of MYC, leading to down-regulation of MYC necessary for alveolar type II cell differentiation. Accumulation in the brain with autosomal-recessive juvenile Parkinsonism, idiopathic Parkinsonism and diffuse lewy body disease.
Exosomes comprising AIMP1 and AIMP2 proteins were prepared by expressing fusion proteins composed of an exosome-specific marker and a target protein in large quantities in high-concentration exosome-producing cells. Exosomes loaded with either AIMP1 or AIMP2 proteins can be used for multifunctional regulation.
Fluorescent protein (mCherry, GFP)
Fluorescent proteins are members of a structurally homologous class of proteins that have the unique property of being able to form themselves chromophores of visible wavelength from 3 amino acid sequences within their own polypeptide sequence. It is common research practice for biologists to introduce genes (or gene chimeras) encoding engineered fluorescent proteins into living cells, followed by observing the location and dynamics of the gene products using fluorescence microscopy.
The most common use of fluorescent proteins is to image the localization and dynamics of specific organelles or recombinant proteins in living cells using fluorescent proteins. To image a particular organelle, standard molecular biology techniques are used to fuse a gene encoding a fluorescent protein to a cDNA encoding a protein or peptide known to be localized to that particular organelle. This fusion allows the chimeric gene to be expressed as a single polypeptide, forming a covalent linkage between the targeting motif and the fluorescent protein. Plasmids containing the chimeric gene under the control of a suitable promoter are used to transfect mammalian cells and the gene is then expressed to produce the corresponding chimeric protein. The chimeras localize to the target organelle and thus exhibit fluorescence. By using fluorescence microscopy, the morphology, kinetics and distribution of organelles can be imaged over time.
mCherry is a monomeric fluorescent structure with peak excitation/emission at 587nm/610nm, respectively. It is resistant to photobleaching and stable. It matures rapidly，t0.515 minutes, which allows it to be visualized shortly after translation.
Green Fluorescent Protein (GFP) is a protein consisting of 238 amino acid residues (26.9kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein that was first isolated from victoria multiphoton jellyfish (Aequorea victoria). GFP from victoria multiphoton luminescent jellyfish (a. victoria) has a major excitation peak at 395nm wavelength and a minor excitation peak at 475 nm. Its emission peak is located at 509nm, in the lower green part of the visible spectrum.
The present invention prepares exosomes carrying mCherry or GFP proteins and demonstrates that the mCherry or GFP protein is delivered to the cytosol of the target cell. The results indicate that exosomes of the present invention loaded with mCherry or GFP proteins can be used for localized and dynamic imaging of exosomes and connexins in living cells or animals.
Nucleic acid binding proteins (e.g., RNP)
A nucleoprotein is any protein structurally related to a nucleic acid, whether DNA or RNA. Deoxyribonuclein (DNP) is a complex of DNA and protein. A typical example is the nucleosome complex, where genomic DNA is surrounded on clusters of eight histones in the eukaryotic nucleus to form chromatin. During spermatogenesis, protamine replaces histone. The deoxyribonucleotides in such complexes interact to produce a multiprotein regulatory complex in which the intervening DNA is surrounded or intertwined. Deoxyribonucleoproteins are involved in regulating DNA replication and transcription.
Ribonucleoproteins (RNPs) are complexes of RNA and protein. Telomerase, fornix ribonucleoprotein, ribonuclease P, hnRNP and micronucleus RNP (snRNP) and ribosomes are ribonucleoproteins. The ribonucleoprotein plays a protective role. mRNA does not exist as free RNA molecules in the cell. They always bind to ribonucleoproteins and function as ribonucleoprotein complexes.
Exosomes comprising DNP or RNP are prepared by expressing in large quantities a fusion protein consisting of an exosome-specific marker and a target protein in high concentration exosome-producing cells. Exosomes carrying DNPs or RNPs may be used for genetic regulatory or nucleic acid-transportable exosomes.
The present invention demonstrates successful delivery of a cargo protein to the cytosol of a target cell by using exosomes comprising the cargo protein, and thus the present invention provides a method of treating disease using exosomes to effectively modulate intracellular signal transduction in the cytosol.
It is another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammatory diseases comprising exosomes as an active ingredient.
It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, comprising exosome as an active ingredient.
It is another object of the present invention to provide a pharmaceutical composition for preventing or treating oxygen-related diseases, comprising exosomes as an active ingredient.
It is another object of the present invention to provide a composition for generating a conditional knock-out allele of a target gene comprising an exosome as an active ingredient.
It is another object of the present invention to provide a composition for manipulating a DNA sequence comprising exosomes as an active ingredient.
It is another object of the present invention to provide a pharmaceutical composition for preventing or treating gaucher disease comprising exosome as an active ingredient.
In order to develop an efficient method for preparing exosomes comprising a cargo protein, the present inventors made various attempts. During our study, the inventors noted exosome-specific markers (CD9, CD63, CD81 and CD 82). These markers belong to the tetraspanin family and are typically 4-fold penetrating membrane proteins. The inventors predict that when a cargo protein is conjugated to a membrane protein of an exosome, the cargo protein will be included relatively easily inside the exosome.
By expressing a fusion protein composed of a cargo protein and an exosome-marker that is abundant particularly on an exosome membrane and can penetrate the cell membrane in a high-concentration-producing exosome cell, exosomes containing the cargo protein can be produced in large quantities.
In particular, the method for preparing exosomes comprising the cargo protein of the present invention is characterized by introducing a polynucleotide encoding a fusion protein consisting of an exosome-specific marker and the cargo protein into an exosome-producing cell.
At this point, in the prepared exosomes, the cargo protein is fused to an exosome-specific marker embedded in the exosome membrane.
In order to solve the problem that the cargo protein is bound to the membrane protein of exosome and is not separated even after reaching the target cell, various attempts have been made, and thus a technique for preparing exosome containing the cargo protein by temporarily conjugating the cargo protein to a marker protein has been developed. For example, light-specific binding proteins such as CIBN and CRY2 may be used herein. In particular, CIBN is expressed as a fusion with CD9, one of the marker proteins. Meanwhile, a gene encoding a fusion protein of CRY2 and the cargo protein was introduced into the exosome-producing cell. Due to CD9, the expressed CIBN-CD9 fusion protein may be included in an exosome-producing cell. At this time, when the cells were irradiated with blue LED light, CRY2 domain of the cargo protein-CRY 2 fusion protein expressed in the exosome-producing cells bound to the CIBN domain fused with CD 9. The result was a reversible "cargo protein-CRY 2-CIBN-CD9 fusion protein". Due to CD9, such a fusion protein may be included inside the exosomes. Once exosomes containing the cargo protein were produced and blue LED light illumination was terminated, the CIBN-CRY2 junction was disrupted and the cargo protein remained in exosomes in a form separated from the exosome membrane, resulting in the preparation of exosomes including the cargo protein (fig. 5-10).
Such exosomes prepared by the method of the present invention are completely different in effect from conventional exosomes containing a target substance. Conventional exosomes are expressed as fused to exosome-specific markers so as to present the cargo protein inside the exosome, such that the cargo protein is not free, even if included inside the exosome, but is present attached to the exosome membrane, indicating that the cargo protein cannot be detached from the exosome membrane, and therefore the cargo protein can be delivered into the target cell only when the exosome is fused to the cell wall of the target cell. Furthermore, even after fusion to a target cell, the cargo protein is conjugated to the exosome membrane, and therefore, the cargo protein has a low possibility of exhibiting its effect in the target cell, however, the exosome of the present invention presents a cargo protein that exists free and is not conjugated to the exosome membrane. Thus, when such exosomes enter the cytosol by endocytosis of the target cell, the cargo protein does not adhere to the membrane of the exosome, and when the exosome is decomposed in the target cell, the cargo protein contained can be delivered and freely move in the cytosol, indicating that the cargo protein has full activity of its physiological activity in the cytosol of the target cell (fig. 11).
The level of binding of the cargo protein to the marker protein may vary depending on the intensity of the light to be irradiated. Thus, by adjusting the intensity of the light, the concentration of the cargo protein collected in the exosomes can be controlled.
The method of preparing exosomes comprising cargo proteins using light-specific binding proteins has not been reported and was first proposed by the present inventors.
In particular, the method of preparing exosomes comprising cargo proteins of the present invention consists of the following steps: (a) introducing a polynucleotide encoding a fusion protein (fusion protein I) and a polynucleotide encoding a fusion protein (fusion protein II) into an exosome-producing cell, wherein the fusion protein I consists of an exosome-specific marker and a first light-specific binding protein, and the fusion protein II consists of a cargo protein and a second light-specific binding protein which can be connected with the first light-specific binding protein; (b) illuminating the exosome-producing cell with light capable of causing conjugation between a first light-specific binding protein and a second light-specific binding protein; and (c) terminating irradiation after completion of exosome production in the exosome-producing cells.
The term "exosome" in the present invention denotes a vesicle having a plasma membrane structure, which originates from a specific compartment within a cell called a multivesicular body (MVB) and is released or secreted from the cell.
In the present invention, exosomes as vectors deliver cargo proteins into target cells or tissues by carrying the cargo proteins themselves. At this time, the cargo protein carried by the exosome acts on the target cell or tissue to help treat or diagnose the specific disease.
The term "exosome-producing cell" in the present invention means a cell capable of producing exosomes.
In the present invention, the exosome-producing cell is not limited, but is preferably exemplified by B lymphocytes, T lymphocytes, dendritic cells, megakaryocytes, macrophages, stem cells, tumor cells, and the like. For example, in the present invention, HEK293T cells used as exosome-producing cells are an immortalized cell line.
The term "exosome-specific marker" in the present invention denotes an exosome membrane-rich protein.
In the present invention, the exosome-specific marker is not limited, but CD9, CD63, CD81, CD82 and the like are preferably exemplified. For example, in a preferred embodiment of the invention, CD9 is used as an exosome-specific marker. CD9, CD63, CD81, and CD82 are 4-fold penetrating membrane proteins that allow the cargo protein to be readily present in exosomes when the cargo protein binds to the membrane protein of the exosomes.
The term "light-specific binding protein" in the present invention, also referred to as a light-induced heterodimer forming protein or a light-induced homodimer forming protein, refers to a protein capable of forming a heterodimer by binding to a different protein or a homodimer by binding to another protein of the same type when light of a specific wavelength is irradiated.
In the present invention, the light-specific binding protein is not limited, but preferably exemplified by light-induced heterodimer forming protein or CIB (cryptochrome-interacting base-helix-loop-helix protein), CIBN (N-terminal domain of CIB), PhyB (phytochrome B), PIF (phytochrome interacting factor), FKF1 (flavin-binding, Kelch repeat, F-box 1), GIGANTEA, CRY (cryptochrome), and PHR (plant hydrolase homologous region), etc.
In particular, when the light-specific binding protein is a light-induced heterodimer forming protein, two types of light-specific binding proteins (first and second light-specific binding proteins) may be used. When the first light-specific binding protein is CIB or CIBN, the second light-specific binding protein may be CRY or PHR. When the first light-specific binding protein is PhyB, the second light-specific binding protein can be PIF. When the first light-specific binding protein is GIGANTEA, the second light-specific binding protein can be FKF 1.
For example, in a preferred embodiment of the invention, CIBN is used as the first light-specific binding protein and CRY2 is used as the second light-specific binding protein. The light used here has a wavelength of 460 to 490nm blue light. The intensity of the light is 20 to 50 μ W.
Meanwhile, in order to confirm the expression of the first fusion protein composed of the exosome-specific marker expressed therein and the first light-specific binding protein and find its position, the marker protein may be fused into the fusion protein. For example, in a preferred embodiment of the invention, the fluorescent protein EGFP is inserted into a first fusion protein in which CIBN and CD9 or GIGANTEA and CD are linked together. Thus, the expression pattern (expression and expression level) and the intracellular location of the first fusion protein can be studied by expression of the first fusion protein carrying the fluorescent protein EGFP.
The term "cargo protein" in the present invention denotes a protein expressed as a fusion protein conjugated to a second light-specific binding protein to localize the cargo protein within exosomes.
In the present invention, the cargo protein may be carried by exosomes after expression in the cell. The cargo protein is not limited, but is preferably a disease therapeutic protein or a disease diagnostic protein. For example, in a preferred embodiment of the invention, mCherry with fluorescence is used as the cargo protein.
Examples of cargo proteins of the present invention are selected from, but not limited to, Matrix Metalloproteinase (MMP) proteins, metalloproteinase Tissue Inhibitor (TIMP) proteins, caspase-inhibiting proteins, cathepsin proteins, or cathepsin-inhibiting proteins.
Wherein,
MMPs proteins such as, but not limited to, MMP1 protein (SEQ ID: 13);
TIMPs proteins such as, but not limited to, TIMP1 protein (SEQ ID NO: 14), TIMP2 protein (SEQ ID NO: 15), TIMP3 protein (SEQ ID NO: 16) or TIMP4 protein (SEQ ID NO: 17);
caspase proteins such as, but not limited to, caspase 1 protein (SEQ ID NO:18), caspase 2 protein (SEQ ID NO:19), caspase 3 protein (SEQ ID NO:20), caspase 4 protein (SEQ ID NO:21), caspase 5 protein (SEQ ID NO:22), caspase 6 protein (SEQ ID NO:23), caspase 7 protein (SEQ ID NO:24), caspase 8 protein (SEQ ID NO:25), caspase 9 protein (SEQ ID NO:26), caspase 10 protein (SEQ ID NO:27), caspase 11 protein (SEQ ID NO:28), caspase 12 protein (SEQ ID NO:29), caspase 13 protein (SEQ ID NO:30) or caspase 14 protein (SEQ ID NO: 31);
caspase inhibitors such as, but not limited to, those that inhibit the expression of a protein represented by SEQ ID NO: 18-31 or any caspase inhibiting protein.
Cathepsin proteins such as, but not limited to, cathepsin A protein (SEQ ID NO:32), cathepsin B protein (SEQ ID NO:33), cathepsin C protein (SEQ ID NO:34), cathepsin D protein (SEQ ID NO:35), cathepsin E protein (SEQ ID NO:36), cathepsin F protein (SEQ ID NO:37), cathepsin G protein (SEQ ID NO:38), cathepsin H protein (SEQ ID NO:39), cathepsin K protein (SEQ ID NO:40), cathepsin L1 protein (SEQ ID NO:41), cathepsin L2 protein (SEQ ID NO:42), cathepsin O protein (SEQ ID NO:43), cathepsin S protein (SEQ ID NO:44), cathepsin W protein (SEQ ID NO:45) or cathepsin Z protein (SEQ ID NO: 46); and
cathepsin inhibiting proteins such as, but not limited to, cathepsin inhibiting proteins represented by SEQ ID NOS: 32-46 or any cathepsin inhibiting protein.
Another example of a cargo protein of the invention is selected from, but not limited to, Cre recombinase, Cas protein, caspase-activated DNase (CAD) protein, β -Glucocerebrosidase (GBA), p38 mitogen-activated protein kinase, phosphatase and tensin homolog (PTEN), Janus kinase (JAK), ubiquitin ligase, luciferase, peroxide oxidoreductase (Prx) I or II, NF-. kappa.B inhibitory protein, MyoD protein, Tbx18 protein, p53 protein, high mobility group protein 1(HMGB1) protein, neurogenic differentiation 1(Neuro-D1) protein, interferon regulatory factor 5(IRF5) protein, interferon regulatory factor 3(IRF3) protein, signal transduction and transcriptional activation 1(STAT1) protein, cytokine signal suppressor 3(SOCS3) protein, signal transduction and transcriptional activation 2(STAT2) protein, STAT inhibitor protein (3) protein, Bax related protein (Bcx 24), BcMX-related lymphokine kinase binding protein (mCHP), protein, or protein, mCHP-binding protein, or protein,
wherein,
cre recombinase recombines DNA between LoxP sites by recognizing LoxP sites in the DNA, including but not limited to DNA represented by SEQ ID: 9, a Cre recombinase represented by;
when the Cas protein is bound to the complex by the guide RNA, it has endonuclease or nickase activity. In some embodiments, the Cas protein is a Cas9 protein (such as the Cas protein represented by SEQ ID NO: 10), or a mutant thereof or a Cpf1 protein (such as the amino acids represented by SEQ ID NO: 11);
CAD proteins are such as those represented by SEQ ID NO: 47 represents an amino acid;
β -Glucocerebrosidase (GBA) such as the amino acid represented by SEQ ID NO: 12;
the p38 mitogen-activated protein kinase (p38MAPKs) protein is, for example, p38- α or a variant thereof, and includes the amino acids represented by SEQ ID NOS: 48-51;
the inhibitor kappa B kinase (IKK) protein is such as is represented by SEQ ID NO: 83 is an amino acid;
nuclear factor-kappaB (NF- κ B) protein such as the polypeptide set forth in SEQ ID NO: 84;
phosphatase and tensin homologous Proteins (PTENs) such as those represented by SEQ ID NO: 52 or an amino acid represented by;
janus kinase (JAK) proteins include JAK1, JAK2, JAK3, and TYK2, wherein JAK1 proteins are such as those identified as SEQ id nos: 53, JAK2 protein such as the amino acid sequence represented in SEQ ID NO: 54, JAK3 protein such as the amino acid sequence represented in SEQ ID NO: 55, such as the amino acid sequence represented in SEQ ID NO: 56; ubiquitin ligase proteins include c-CBL, PRKN, RBX1, TRAF2 and Mdm2, wherein ubiquitin ligase proteins such as the amino acid sequences set forth in SEQ ID NO: amino acids represented by 57 to 61;
luciferase proteins such as those represented by SEQ ID NO: 62 or an amino acid represented by;
the peroxiredoxin (Prx) I or II, such as a peroxiredoxin represented by SEQ ID NO: 7, oxidase II such as is represented by SEQ id no: 8 represents an amino acid;
NF-. kappa.B inhibitory proteins are the supersuppressor IkB which inactivates NF-. kappa.B by binding to NF-. kappa.B in the cytoplasm, wherein the supersuppressor IkB protein (S32A and S36A mutant forms of IkB) is not phosphorylated by IkB kinase (IKK), and therefore it can continuously inhibit NF-. kappa.B, and NF-. kappa.B inhibitory proteins such as amino acids represented by one of SEQ ID NOS: 1 to 5, e.g., IkB-. alpha., IkB-. beta., IkB-. epsilon., BCL-3 or mutants thereof;
the MyoD protein is such as is represented by SEQ ID NO: 63;
the Tbx18 protein is such as is represented by SEQ ID NO: 64 represents an amino acid;
the P53 protein is such as is represented by SEQ ID NO: 65, or a pharmaceutically acceptable salt thereof;
high mobility group box 1 protein (HMGB1) is represented by SEQ ID NO: 66 or an amino acid represented by;
a neurogenic differentiation 1(Neuro-D1) protein such as is represented by SEQ ID NO: 67 represents an amino acid;
an interferon regulatory factor 5(IRF5) protein such as is represented by SEQ ID NO: 68;
an interferon regulatory factor 3(IRF3) protein such as is represented by SEQ ID NO: 69;
a signal transducer and activator of transcription 1(STAT1) protein such as is represented by SEQ ID NO: 70 or a pharmaceutically acceptable salt thereof;
a cytokine signaling inhibitor 3(SOCS3) protein such as is represented by SEQ ID NO: 71;
a signal transducer and activator of transcription 2(STAT2) protein such as is represented by SEQ ID NO: 72, or an amino acid represented by;
proteins that inhibit phosphorylated STAT3 (pySTAT3), including pySTAT3 intrabody proteins that bind to pySTAT3 to inactivate pySTAT3, and such as the one or more of SEQ ID NO: 73 or any protein that inhibits pySTAT 3;
bax (Bcl 2-related X protein) is exemplified by the EQ ID NO: 6 represents an amino acid;
b cell lymphoma-extra large (Bcl-xL) proteins such as those represented by SEQ ID NO: 74;
aminoacyl tRNA synthetase interacting multifunctional proteins (AIMPs) include AIMP1 and AIMP2, wherein the AIMP1 protein is, for example, an amino acid sequence as defined in EQ ID NO: 75 and AIMP2 protein such as a peptide represented by SEQ ID NO: 76;
the mCherry protein is, for example, represented by EQ ID NO: 77;
green Fluorescent Protein (GFP) is exemplified by the amino acid sequence as EQ ID NO: 78 with a substituent;
nucleic acid-binding nucleoproteins include DNA-binding Deoxyribonucleoproteins (DNPs) or RNA-binding Ribonucleoproteins (RNPs), wherein DNPs include RBBP4 or NAP1L4, and RNPs include telomerase, heterologous ribonucleoprotein K (HNRNPK), and wherein nucleoproteins such as nucleosomes, protamines, micronucleus RNPs (snrnps), the coding sequence set forth in SEQ ID NO: 79-82 or a mutant thereof or any protein which binds to nucleic acids.
The term "culturing" in the present invention means a method of growing cells or microorganisms in a suitably controlled environment.
In the invention, the transformant is cultured for 1-3 days, then the culture medium is changed into a serum-free culture medium, and the culture is continued for 2-5 days.
In the present invention, the method for culturing the transformant is any method known to those skilled in the art.
The medium herein refers to a notification medium widely used for animal cell culture, and may be selected from the group consisting of commercially available serum-free medium, protein-free medium, and chemically-defined medium.
The serum-free medium described above was used for animal Cell culture, without bovine serum and exemplified by SFM4CHO (HyClone) and EX-Cell (JHR Bioscience). Insulin-like growth factor I (IGF-I), ethanolamine, ferric chloride, and phosphatidylcholine may be added to the medium, but are not always limited thereto.
The above protein-free medium is an animal cell medium from which animal-derived proteins (particularly high molecular weight proteins, particularly proteins having a molecular weight of at least 10 kDa) are removed, and may be ProCHO (Lonza) and PF-Cho (HyClone), but is not always limited thereto.
The chemically defined medium described above is an animal cell culture medium which does not contain any animal-derived components, but has all chemically defined components. The chemically-defined medium may be CDM4CHO (HyClone), PowerCHO2CD (Lonza) and CD-optiCHO (Life Technologies), but is not always limited thereto.
The term "first fusion protein" in the present invention denotes a fusion protein formed by binding of an exosome-specific marker to a first light-specific binding protein.
In the present invention, the order of arrangement of the exosome-specific marker and the first optical specific binding protein comprised in the first fusion protein is not limited as long as the first photosensitive specific binding protein is located in a direction toward the inside of an exosome when the first fusion protein is expressed in an exosome-producing cell. For example, the N-terminus of the first light-specific binding protein may be conjugated to the C-terminus of an exosome-specific marker.
The exosome-specific marker and the first light-specific binding protein constituting the first fusion protein are directly linked to each other or may be linked by a linker, which is not limited as long as the first fusion protein is expressed in an exosome-producing cell and presents the first light-specific binding protein toward the inside of the exosome, but is preferably a peptide linker composed of amino acids, and more preferably a flexible peptide linker. The peptide linker may be expressed by using an expression vector in which the nucleic acid encoding the linker is linked to other nucleic acids encoding the respective domains in the framework.
The term "second fusion protein" denotes a fusion protein in which the second light-specific binding protein and the cargo protein are bound.
In the present invention, the order of arrangement of the second light-specific binding protein and the cargo protein contained in the second fusion protein is not limited as long as the second fusion protein is located within the exosome and binds to the first light-specific binding protein region of the first fusion protein in the exosome-producing cell. For example, the N-terminus of the cargo protein can be conjugated to the C-terminus of the second light-specific binding protein.
The second light-specific binding protein and the cargo protein constituting the second fusion protein are directly linked to each other or may be linked by a linker, which is not limited as long as the second fusion protein is located within the exosome and binds to the first light-specific binding protein region of the first fusion protein in the exosome-producing cell, but is preferably a peptide linker composed of amino acids, and more preferably a flexible peptide linker. The peptide linker may be expressed by using an expression vector in which the nucleic acid encoding the linker is linked to other nucleic acids encoding the respective domains in the framework.
In addition, each of the above fusion proteins may include a polypeptide having a sequence in which at least one amino acid residue differs from an amino acid residue in the wild-type amino acid sequence of each domain included in each of the fusion proteins. Amino acid exchanges in proteins and polypeptides without altering the overall activity of the molecule are well known to those skilled in the art. The most common exchanges occur at Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly. In addition, proteins having improved structural stability to heat or pH or increased protein activity due to mutation or modification of amino acid sequences may be included.
Finally, the above-described fusion protein or polypeptide including each domain fusion protein can be prepared by a chemical peptide synthesis method well known to those skilled in the art, or by the following method. The genes encoding the respective domains are amplified by PCR (polymerase chain reaction) or synthesized by conventional methods well known to those skilled in the art. The gene was cloned into an expression vector and expressed.
Meanwhile, each fusion protein may be expressed in an exosome-producing cell by introducing a polynucleotide encoding each fusion protein into the exosome-producing cell. At this time, the polynucleotide is introduced into the exosome-producing cell by a conventional method well known to those skilled in the art. For example, expression vectors can be used for introduction.
The term "expression vector" in the present invention is a recombinant vector capable of expressing a target peptide in a host cell. The vector refers to a genetic construct comprising key regulators operably linked to express a gene insert. Expression vectors include expression control elements such as start codons, stop codons, promoters and operators. The start codon and stop codon are generally understood as being part of the nucleotide sequence encoding the polypeptide. Gene constructs are considered to be functional when they are introduced and present in the coding sequence in frame. The promoter of the vector may be constitutive or inducible.
The term "operably linked" in the context of the present invention refers to the state when the ordinarily functioning nucleic acid expression regulatory sequence and the nucleic acid sequence encoding the cargo protein or RNA are linked by a functional linkage. For example, a promoter is operably linked to a nucleic acid sequence encoding a protein or RNA to affect expression of the coding sequence. Functional linkage to an expression vector can be achieved by recombinant DNA techniques well known to those skilled in the art, and in particular site-specific DNA cleavage and linkage can be achieved by using conventional enzymes well known to those skilled in the art.
The expression vector may include a signal sequence for export of the fusion polypeptide to facilitate isolation of the protein from the cell culture medium. Specific initiation signals may be necessary for efficient translation of the inserted nucleic acid sequence. These signals contain the ATG initiation codon and its adjacent sequences. In some cases, exogenous translational control signals, which may include an ATG initiation codon, should be provided. These exogenous translational control signals and initiation codons can be of various natural and synthetic origins. The expression efficiency can be increased by introducing an appropriate transcription enhancer or translation enhancer.
In a preferred embodiment of the invention, the expression vector is capable of expressing the cargo protein conjugated to the tag to confirm insertion of the cargo protein within the exosome. The tags herein are used to confirm the presence of a cargo protein that can be conjugated to a region opposite to the region to which the second light-specific binding protein is conjugated. For example, fluorescent proteins such as red fluorescent protein and green fluorescent protein are used as tags conjugated to the C-terminus of the cargo protein.
The cargo protein prepared as described above is expressed in an exosome-producing cell. Once the exosomes are generated, it is investigated whether a fluorescent protein tag is detected, from which the presence of cargo protein in the exosomes can be confirmed.
The term "light" in the present invention means light to be irradiated so as to temporarily bind a first light-specific binding protein and a second light-specific binding protein expressed in an exosome-producing cell.
As described above, the first light-specific binding protein is expressed as a first fusion protein conjugated to an exosome-specific marker, while the second light-specific binding protein is expressed as a second fusion protein conjugated to a cargo protein. When light is irradiated to the exosome-producing cell, the first photo-specific binding protein binds to the second photo-specific binding protein, and as a result, a fusion protein complex comprising exosome-specific marker-first photo-specific binding protein-second photo-specific binding protein-cargo protein is transiently formed. When exosomes are produced in exosome-producing cells, the cargo protein may be linked to the exosome due to an exosome-specific marker. At this time, the cargo protein exists within the exosome, and when the light irradiation is stopped after the exosome is produced, the first light-specific binding protein is separated from the second light-specific binding protein, and thus the cargo protein included in the exosome is discharged together with the exosome as a part of the exosome. To more efficiently deliver the cargo protein into the exosomes, it is preferred to irradiate the cell with light intermittently rather than continuously. That is, when light is intermittently irradiated, the conjugation and separation of the first light-specific binding protein and the second light-specific binding protein are repeated, so that the possibility of introduction of the cargo protein into exosomes can be increased.
At the same time, the wavelength of light sufficient to induce binding of the first light-specific binding protein to the second light-specific binding protein varies depending on the species of the first and second light-specific binding proteins. The wavelength of light that induces binding of the first light-specific binding protein and the second light-specific binding protein depends on the type of protein. Thus, as known to those skilled in the art, an appropriate wavelength of light may be selected. For example, in order to connect CRY2 to CIBN, light having a wavelength of 460 to 490nm is preferable. If the light is irradiated for less than 10 minutes, CRY2 and CIBN are separated from each other. When PhyB was combined with PIF, light was irradiated for 10 minutes at a wavelength of 650 nm. When irradiated with light having a wavelength of 750nm for 5 minutes, PhyB and PIF were separated from each other. When FKF1 was bound to GIGANTEA, it was irradiated with light having a wavelength of 460nm for 30 minutes. In a preferred embodiment of the present invention, the combination of CIBN and CRY2 is induced by irradiating with light having a wavelength of 460 to 490 nm.
In a preferred embodiment of the invention, the CRY2/mCherry fusion protein and the CIB/CD9 fusion protein are expressed in HEK293T and the immortalized cell line produces a large number of exosomes. As a result, it was found that mCherry protein uniformly distributed in cytosol was distributed in cell membrane and endosome-like structural membrane upon blue light irradiation (fig. 7). Similar results were observed when FKF1/mCherry fusion protein and GIGANTEA/CD9 fusion protein were expressed in HEK293T cells (FIG. 12). The CRY2/mCherry fusion protein and the CIBN/CD9 fusion protein were expressed in HEK293T cells, followed by irradiation with blue light and modulation of light intensity. As a result, the level of mCherry protein collected in exosomes was highest when irradiated with light at intensity positions 20-50 μ W (FIG. 9). Exosomes isolated from cells were allowed to act at a concentration of approximately 250 μ g/ml on HT1080 cells. As a result, the exosomes did not show any specific cytotoxicity to HT1080 cells and demonstrated that the mCherry protein was delivered in its cytosol (fig. 10).
To compare the efficiency of introduction of the cargo protein into exosomes and the efficiency of exosome transfer to target cells in the conventional methods, XPACK vector was used in the conventional method and expression vectors for CRY2/mCherry fusion protein and CIBN/CD9 fusion protein were introduced into HEK293T cells. Then, production of cargo protein in exosomes was compared. The results confirmed that the introduction efficiency was significantly high when the method of the present invention was used (fig. 15). Exosomes isolated from exosome-producing cells were acted on target cells (HeLa) to compare the expression of cargo proteins. When exosomes isolated by the method of the present invention were used, the expression of the cargo protein was highest in the target cells (figure 16).
In another preferred embodiment of the present invention, the present invention provides a vector for producing exosomes, comprising: (a) a first expression vector comprising a polynucleotide encoding a fusion protein of an exosome-specific marker and a first light-specific binding protein (first fusion protein); and (b) a second expression vector comprising a multiple cloning site and a polynucleotide encoding a second light-specific binding protein linked to the first light-specific binding protein, wherein a polynucleotide encoding a cargo protein can be introduced into the multiple cloning site.
In the vector for producing exosomes provided by the present invention, the exosome-specific marker, the first light-specific binding protein, the exosome-producing cell and the second light-specific binding protein are the same as described above.
In the present invention, the term "transformed cell for exosome production" denotes a cell capable of producing exosomes by expressing a first fusion protein, into which a polynucleotide encoding a fusion protein of an exosome-specific marker and a first light-specific binding protein (first fusion protein) is introduced.
In the present invention, the second expression vector comprises a polynucleotide encoding a second light-specific binding protein and an adjacent multiple cloning site. When the polynucleotide encoding the cargo protein is inserted into the multiple cloning site, it is expressed as a fusion protein comprising the second light-specific binding protein and the cargo protein (second fusion protein).
The vector for preparing exosomes provided by the present invention may comprise one or more of components, solutions or devices, which are useful not only for the transformed cells and expression vectors for producing exosomes, but also for introducing expression vectors; for culturing transformed cells producing exosomes; and for isolating and purifying exosomes produced from exosome-producing transformed cells. For example, a buffer suitable for introduction of the expression vector and a medium and a vessel necessary for culturing the transformant cell producing the exosome may also be included.
The term "Cas protein" in the present invention denotes a key protein in a CRISPR/Cas system, which forms an active endonuclease or nickase when the Cas protein forms a complex with two RNAs called CRSPR RNA (crRNA) and trans-activating crRNA (tracrRNA).
The term "guide RNA" in the present invention indicates a target-DNA specific RNA capable of forming a complex with a Cas protein and guiding the Cas protein to a target DNA.
In the present invention, the guide RNA may be prepared from two RNAs, CRSPRRNA (crRNA) and trans-activating crRNA (tracrRNA) or single-stranded RNA (SgRNA), by fusing essential parts of crRNA and tracrRNA.
The guide RNA may be a double RNA (daul RNA) including crRNA and tracrRNA. Any guide RNA can be used in the present invention if the above RNA includes the essential and target complementary portions of crRNA and tracrRNA. The crRNA can hybridize to the target DNA.
The guide RNA can include one or more additional nucleotides at the 5 'end of the single stranded guide RNA or the 5' end of the crRNA in the duplex RNA.
Ideally, the guide RNA can include two additional guanine nucleotides at the 5 'end of the single stranded guide RNA or the 5' end of the crRNA in the duplex RNA. The guide RNA can be delivered to the cell or organism as RNA or DNA encoding the guide RNA. The guide RNA is RNA that can be isolated, RNA contained in a viral vector, or encoded in a vector. Desirably, the above vector is not limited, but may be a viral vector, a plasmid vector or an agrobacterium vector.
The DNA encoding the guide RNA may be a vector including a DNA sequence encoding the guide RNA. For example, the guide RNA can be delivered to the cell or organism by transfection with plasmid DNA that includes an isolated guide RNA or a sequence encoding a guide RNA and a promoter. According to other methods, the guide RNA may be delivered to the cell or organism by using virus-mediated gene delivery.
When the guide RNA is transfected into a cell or organism as isolated RNA, the guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the industry. Ideally, the guide RNA is delivered to the cell in the form of an isolated RNA rather than in the form of a plasmid comprising a sequence encoding the guide RNA. In the present invention, the term "isolated RNA (isolated RNA)" may be replaced by "naked RNA (naked RNA)". Since the isolated RNA does not require a cloning process, costs and time can be saved. However, the use of plasmid DNA or virus-mediated gene delivery for guiding RNA transfection is not excluded.
The invention provides exosomes comprising cargo proteins prepared by the methods of the invention.
In another aspect, the present invention provides an exosome comprising a cre recombinase produced by the above-described method.
In another aspect, the invention provides an exosome comprising a Cas9 protein prepared by the above method.
in another aspect, the present invention provides an exosome comprising a GBA (β -glucocerebrosidase) protein produced by the above-described method.
In another aspect, the invention provides exosomes comprising a peroxide oxidoreductase (Prx) I or II protein produced by the above-described method.
In another aspect, the present invention provides exosomes comprising a protein that inhibits NF-kB produced by the above method.
In another aspect, the present invention provides an exosome comprising a Bax (Bcl-2 associated X protein) protein prepared by the above-described method.
The exosomes prepared by the above method comprise a fusion protein on an exosome membrane (first fusion protein) consisting of an exosome-specific marker and a first light-specific binding protein and another fusion protein (second fusion protein) consisting of a cargo protein and a second light-specific binding protein that can be conjugated to the first light-specific binding protein. Thus, when such exosomes are acted on a target tissue cell, the second fusion protein contained in the exosomes may be delivered to the cytosol of the target tissue cell through plasma membrane fusion.
The exosomes comprising the cargo protein are useful for the treatment of various diseases in vivo. For example, exosomes containing protein polymers (e.g., antibodies, etc.) exhibiting anticancer activity as cargo proteins are prepared and then allowed to act on cancer cells. That is, exosomes can be used as biocompatible anticancer agents that function better than conventional liposomes.
The present invention also provides pharmaceutical compositions for the prevention and treatment of inflammatory diseases, including exosomes having NF- κ B inhibitory proteins.
The inflammatory disease is not limited, but is preferably allergy, dermatitis, idiopathy, conjunctivitis, periodontitis, rhinitis, otitis media, pharyngolaryngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, scapulohumeral periarthritis, tendonitis, tenosynovitis, peritendonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, acute and chronic inflammatory disease, sepsis, ulcerative colitis, and the like.
in the experimental example of the present invention, the present inventors confirmed that HeLa cells were pretreated with the super repressor IkB: EXPLOR to inhibit the transfer of NF-kB activated by TNF- α to the nucleus, to confirm the inflammation inhibitory effect mediated by TNF- α (FIG. 43. left). Inaddition, it was confirmed that DNA binding of TNF- α activated NF-kB was inhibited (FIG. 43 right). Inaddition, the present inventors confirmed that the symptoms of arthritis were alleviated in a mouse model in which collagen was injected 3 times after eye to induce arthritis, to confirm the inflammation inhibitory effect, and thus the super repressor IkB: EXPLOR of the present invention can be used as a pharmaceutical ingredient for the prevention and treatment of inflammatory diseases.
The present invention also provides pharmaceutical compositions for preventing and treating cancer, including exosomes comprising Bax (Bcl-2-associated X protein).
The above cancers are not limited, but preferred examples are breast cancer, colon cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or choroidal melanoma, eye cancer, peritoneal cancer, uterine cancer, ovarian cancer, rectal cancer, anal cancer, cervical cancer and the like.
In experimental examples of the present invention, the present inventors confirmed that pre-treating HeLa cells with Bax: EXPLOR increases the release of cytochrome c, whereby Bax: EXPLOR can be used as a pharmaceutical component for preventing and treating cancer.
The invention also provides pharmaceutical compositions for use in oxidation resistance, including exosomes comprising peroxide oxidoreductase (Prx) I or II.
In addition, the present invention provides pharmaceutical compositions comprising exosomes containing peroxide oxidoreductase (Prx) I or II for preventing and treating reactive oxygen species diseases, such as cancer, arteriosclerosis, respiratory diseases, osteoporosis, obesity, and degenerative dementia, etc.
In addition, the present invention provides cosmetic compositions for anti-oxidation, including exosomes having peroxide oxidoreductase (Prx) I or II.
In addition, the present invention provides cosmetic compositions for skin anti-aging, including exosomes having peroxide oxidoreductase (Prx) I or II.
In the experimental examples of the present invention, the present inventors confirmed that pretreatment of HeLa cells with Prx I/II EXPLOR statistically significantly inhibited cytotoxicity caused by oxidative stress to verify against H2O2Induces an inhibitory effect on cytotoxicity caused by oxidative stress, and thus PrxI/II EXPLOR can be used as a pharmaceutical ingredient for antioxidation or prevention and treatment of active oxygen, or as a cosmetic ingredient for antioxidation or anti-aging of skin.
The invention also provides a composition comprising an exosome with Cre recombinase for creating a conditional knock-out allele of a target gene.
In the experimental examples of the present invention, the present inventors confirmed the effect of Cre recombinase by detecting expression of ZsGreen reporter protein after transfecting pCAG-loxP-STOP-loxP-ZsGreen encoding DNA into HT1080 and HeLa cells, that expression of ZsGreen reporter protein in EXPLOR-treated HT1080 cells and HeLa cells was the same as that of pCMV-Cre vector transfection as a positive control (FIGS. 19a and 19 b). In addition, the present inventors were able to confirm the expression of ZsGreen on Cre: EXPOR treated primary mouse embryonic neurons by performing the same experiments as described above (FIG. 20). Furthermore, the present inventors confirmed that EYFP was expressed on Cre:: EXPLOR treated group after injecting Cre:: EXPLOR extraabdominally into pCAG-lowP-STOP-loxP-eNPR3.0-EYFP transgenic mice to verify Cre-EXPLOR function in vivo (FIG. 21). Furthermore, it was demonstrated that Cre:: EXPLOR in the immunohistochemical results was targeted to neurons in mouse brain mainly by merging neuronal regions to verify that Cre:: EXPLOR targeted cells (FIG. 22) and thus Cre:: EXPLOR could be used as a component for creating conditional knock-out alleles of target genes.
The invention also provides components for engineering DNA sequences, including exosomes having a Cas9 protein and a target DNA-specific guide RNA (gRNA).
The aforementioned components are not limited, but preferably induce mutations or correct mutations on normal sequences. The mutation may be a naturally occurring mutation or a mutation induced by a pathogenic microorganism. In other words, when a pathogenic microorganism is detected and it is clear that a biological sample is infected, the mutation is caused by infection by the pathogenic microorganism. The pathogenic microorganism is not limited, but may be a virus or a bacterium.
In the experimental examples of the present invention, the present inventors confirmed that exosomes comprising CRISPR/Cas9 proteins can be prepared with high yield.
the invention also provides a pharmaceutical composition for the treatment of gaucher's disease comprising exosomes comprising β -Glucocerebrosidase (GBA).
in the experimental examples of the present invention, the present inventors confirmed that the activity of β -Glucocerebrosidase (GBA) can be restored by treating cells from gaucher's disease patients with GBA:: EXPLOR (FIG. 30), and thus GBA:: EXPLOR can be used as a pharmaceutical ingredient for treating gaucher's disease.
According to the method for producing exosomes comprising a cargo protein of the present invention, exosomes comprising a cargo protein can be produced in high yield. In addition, the cargo protein appears to be separated from the exosome membrane and thus can be widely used for treating diseases.
MODE OF THE INVENTION
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 6: comparison of efficiency of exosome transfer to target cells
To compare exosome transfer efficiencies, target cells were treated with exosomes comprising the cargo protein. In particular, with 5X 109HeLa cells were treated with each exosome for 24 hours, and then the fluorescence intensity expressed in the cells was measured. As shown in fig. 16, it was confirmed that the fluorescence intensity in the Exosomes (EXPLOR) of the present invention was significantly high (fig. 16).
Thus, it was demonstrated that cargo proteins can be more efficiently delivered to target cells using the method of the exosomes of the present invention.
Experimental examples
Experimental example 1: MMP (matrix metalloproteinase)
Binding of CIBN to CRY2 and loading of MMPs in exosomes was evaluated in cells expressing CIBN-EGFP-CD9 and MMPs-mCherry-Cry2 under blue light at 488nm wavelength.
In order to produce large amounts of exosomes loaded with MMPs, cells stably expressing the CIBN-EGFP-CD9 gene and the MMP-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of MMP loaded exosomes in target cells:
target cells were treated with MMP-loaded exosomes to assess functional enzyme activity.
MMP-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 2: TIMP (tissue inhibitor of metalloproteinases)
The binding of CIBN to CRY2 and the loading of TIMPs in exosomes in cells expressing CIBN-EGFP-CD9 and TIMPs-mCherry-Cry2 at blue light wavelength 488nm was evaluated.
In order to produce large amounts of TIMPs-loaded exosomes, cells stably expressing the CIBN-EGFP-CD9 gene and the TIMPs-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of TIMP loaded exosomes in target cells:
target cells were treated with TIMPs loaded exosomes to assess functional enzyme activity.
TIMP-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 3: caspase
Binding of CIBN to Cry2 and caspase-mCherry-Cry 2 in cells expressing CIBN-EGFP-CD9 and caspase-mCherry-Cry 2 at blue light wavelength of 488nm and loading of caspase in exosomes were evaluated.
In order to produce caspase-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the caspase-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by Tangential Flow Filtration (TFF) method.
Functional analysis of caspase-loaded exosomes in target cells:
target cells were treated with caspase-loaded exosomes to assess functional enzyme activity.
Caspase-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic effect.
Experimental example 4: cathepsin (cathepsin)
Binding of CIBN to Cry2 and loading of cathepsin in exosomes in cells expressing CIBN-EGFP-CD9 and cathepsin-mCherry-Cry 2 at blue light wavelength 488nm was evaluated.
In order to produce the cathepsin-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the cathepsin-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of cathepsin loaded exosomes in target cells:
target cells were treated with cathepsin-loaded exosomes to assess functional enzyme activity.
Cathepsin-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic effect.
Experimental example 5: cre recombinant protease
<5-1> production of exosomes loaded with Cre recombinase (Cre:: EXPLOR)
A. Demonstration of Cre recombinase in exosomes
The present inventors demonstrated the binding of CIBN and CRY2 in cells expressing CIBN-EGFP-CD9 and Cre-mCherry-CRY2 to verify that the exosome load has the sequence recorded as SEQ ID NO: 9, Cre recombinase of amino acid. In particular, after 24 hours of culture in the absence of light using a transfected pcDNA3.1(+) vector including the CIBN-EGFP-CD9 gene and the Cre-mCherry-CRY2 gene, the exosome-producing cell HEK293T was cultured in Dulbecco's Modified Eagle's Medium (DMEM) without Fetal Bovine Serum (FBS) for another 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before and after blue light irradiation at wavelength 488nm was investigated by confocal microscopy. This experiment was performed more than five times.
According to the results, the binding between Cre-mCherry-CRY2 (red) and CIBN-EGFP-CD9 was confirmed (FIG. 18), thereby verifying the loading of Cre recombinase by exosomes.
B. Producing an exosome loaded with Cre recombinase (Cre:: EXPLOR)
The present inventors performed the following experiments to obtain an exosome loaded with Cre recombinase.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Cre-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T, and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
In addition, an exosome-producing cell HEK293T which stably expresses a CIBN-EGFP-CD9 gene and a CRE-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. The exosome concentrate was then removed by centrifugation (10000-14000 g, 5 min) on amicon ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min) with a buffer that was preferred according to experimental purpose.
<5-2> confirmation of Cre recombinase function by Cre recombinase-loaded exosomes (Cre:: EXPLOR)
A. Demonstration of Cre:: EXPLOR function on HT1080 and HeLa cells
The Cre recombinase has a function of recombining DNA at the loxP region. The present inventors carried out the following experiment to investigate the function of Cre, EXPLOR.
Specifically, DNA encoded by pCAG-loxP-STOP-loxP-ZsGreen was transfected into HT1080 and HeLa cells and washed after 6 hours. The pCMV-Cre vector is then treated or transfected with 0.25mg/ml Cre:: EXPLOR or negative:: EXPLOR. After 48 hours of culture, the expression of ZsGreen with green fluorescence was studied. Expression of ZsGreen was confirmed in Cre:: EXPLOR treated HT1080 and HeLa cells, which is different from negative:: EXPLOR treated HT1080 and HeLa cells, and was similar to the result of pCMV-Cre vector transfection in the positive control (FIGS. 19a and 19 b).
B. Demonstration of Cre-function of EXPLOR on Primary rat embryonic neurons
The following experiments were performed to study the function of EXPLOR on primary rat embryonic neurons.
Specifically, pCAG-loxP-STOP-loxP-ZsGreen encoding DNA was transfected into primary rat embryonic neurons and washed after 6 hours. They were then cultured on 0.15mg/ml Cre: EXPLOR. After 48 hours of culture, the expression of ZsGreen with green fluorescence was studied. This experiment was performed in at least three replicates, thereby demonstrating the expression of ZsGreen on Cre:. EXPLOR treated primary rat embryonic neurons (FIG. 20).
C. Demonstration of in vivo function of Cre:: EXPLOR in transgenic mice
The present inventors performed the following experiments to verify the function of Cre, EXPLOR, in vivo.
Specifically, 50. mu.l of creEXPLOR (10mg/mL) was injected into pCAG-loxP-STOP-loxP-eNPHR3.0-EYFP transgenic mice by intraperitoneal injection. After injection, 4% formaldehyde fixed brain sections were imaged by fluorescence microscopy. Green fluorescence indicates the expression of eNPR3.0-EYFP, blue fluorescence indicates the nucleus. Expression of eNPH3.0-eYFP on neurons in the Zona Incerta (ZI) of EXPLOR-treated mice was investigated by confocal microscopy and thus confirmed in Cre:EXPLOR-treated pCAG-loxP-STOP-loxP-eNPH3.0-eYFP transgenic mice group (FIG. 21).
D. Demonstration of Cre:. EXPLOR-targeted cells in transgenic mice
Immunohistochemistry experiments were performed in order to confirm the specific cell targeting of EXPLOR in the in vivo experiments described above. Specific staining of neurons with the NeuN antibody and specific staining of astrocytes with the GFAP antibody, and thus confirmed that Cre: EXPLOR specifically targets neurons in mouse brain by studying that the fusion region is mainly neurons (FIG. 22).
Experimental example 6: CRISPR-Cas9
< 6-1 > production of Cas 9-loaded exosomes (Cas9:: EXPLOR)
A. Demonstration of Cas9 in exosomes
The inventors investigated the binding of CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 expressing CIBN and CRY2 to confirm that the amino acid sequence recorded in SEQ ID NO: loading of Cas9 in 10.
As shown in fig. 23, the pcdna3.1(+) vector into which Cas9-mCherry-CRY2 was inserted had a length of 11,890 base pairs, and the three protein portions consisted of Cas9 with an NLS sequence at the 5-terminus, mCherry with a 45 base pair linker sequence, and cryptochrome 2 with 45 and 27 base pair linker sequences, respectively. Each protein portion is 4194, 699 and 1497 base pairs in length, respectively.
Specifically, after 24 hours of culture in the absence of light using a transfected pcDNA3.1(+) vector including the CIBN-EGFP-CD9 gene and the Cre-mCherry-CRY2 gene, the exosome-producing cell HEK293T was cultured in Dulbecco's Modified Eagle's Medium (DMEM) without Fetal Bovine Serum (FBS) for another 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before or after irradiation with blue light of 488nm wavelength was investigated by confocal microscopy. This experiment was performed more than five times. Thus, by confirming that CIBN-EGFP-CD9 binds to Cas9-mCherry-CRY2 under blue light stimulation (fig. 23), Cas9 protein was loaded into exosomes.
B. Generating an exosome loaded with Cas9 (Cas9:: EXPLOR)
The inventors performed the following experiments to obtain Cas 9-loaded exosomes.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Cre-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T, and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
In addition, an exosome-producing cell HEK293T which stably expresses a CIBN-EGFP-CD9 gene and a CRE-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. Then, the liquid in the exosome concentrate was removed by centrifugation (10000-14000 g, 5 min) on Amicon Ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min) with a buffer that was preferred according to experimental purpose. The amount of Cas9 in exosomes was evaluated (fig. 25).
Functional analysis of Cas 9-loaded exosomes in target cells:
target cells were treated with Cas 9-loaded exosomes to show functional enzymatic activity.
Cas 9-loaded exosomes were administered intraperitoneally or intravenously to animal models to demonstrate therapeutic effect.
Experimental example 7: caspase Activated DNase (CAD)
Binding of CIBN to CRY2 and loading of CAD in exosomes was evaluated in cells expressing CIBN-EGFP-CD9 and CAD-mCherry-Cry2 under blue light at 488nm wavelength.
In order to produce CAD-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and CAD-mCherry-CRY2 gene were established and purified exosomes were isolated from culture supernatants by Tangential Flow Filtration (TFF) method.
Functional analysis of CAD-loaded exosomes in target cells:
target cells were treated with CAD-loaded exosomes to show functional activity.
CAD-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 8 β -glucocerebrosidase
< 8-1 > Loading of GBA producing exosomes (GBA:: EXPLOR)
A. Demonstration of GBA in exosomes
The inventors investigated the binding of CIBN to CIBN-EGFP-CD9 and Cas9-mCherry-CRY2 of CRY2 to confirm that the amino acid sequence recorded in SEQ ID NO: the load of the GBA in 12.
[ Table 2]
Specifically, after culturing an exosome-producing cell HEK293T24 with a transfected pcdna3.1(+) vector containing the CIBN-EGFP-CD9 gene and the Cre-mCherry-CRY2 gene under no light conditions for 24 hours, the exosome-producing cell HEK293T was cultured in Dulbecco's Modified Eagle Medium (DMEM) without Fetal Bovine Serum (FBS) for another 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before and after blue light irradiation at a wavelength of 488nm was investigated by confocal microscopy. This experiment was performed more than five times. The GBA protein was thus loaded into exosomes by confirming that CIBN-EGFP-CD9 binds to Cas9-mCherry-CRY2 under blue light stimulation (fig. 26).
B. Production of GBA loaded exosomes (GBA:: EXPLOR)
The present inventors performed the following experiments to obtain GBA-loaded exosomes.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Cre-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T, and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
In addition, an exosome-producing cell HEK293T which stably expresses a CIBN-EGFP-CD9 gene and a CRE-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, cell debris-removed supernatant was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. Then, the liquid in the exosome concentrate was removed by centrifugation (10000-14000 g, 5 min) on amicon ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min) with a buffer that was preferred according to experimental purpose.
Measurement of GBA expression in < 8-2 > GBA-loaded exosome-producing cells
The inventors performed western blotting to determine GBA expression in GBA-loaded exosomes.
specifically, vectors containing the CIBN-EGRP-CD9 gene and the GBA-mCherry-CRY2 gene were transfected onto exosome-producing cells HEK293T and these cells were cultured for 24 hours HEK293T cells were lysed using MPER (mammalian protein extraction reagent) and proteins were analyzed by western blotting rat primary astrocytes, human primary astrocytes and fibroblasts derived from gaucher disease patients, in which β -glucocerebrosidase deficiency was caused due to GBA gene abnormality, were lysed to perform western blotting and proteins were analyzed by western blotting.
As a result, endogenous GBA was observed in HEK293T cells, rat primary astrocytes and human primary astrocytes including CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene, in addition to fibroblasts derived from gaucher disease patients (fig. 27).
In addition, GBA-mCherry-CRY2 fusion protein (151kDa) was observed in HEK293T cells including CIBN-EGRP-CD9 gene and GBA-mCherry-CRY2 gene, indicating that GBA-mCherry-CRY2 fusion protein is well expressed in GBA-loaded exosomes (fig. 28).
< 8-3 > demonstration of GBA Activity on cells derived from gaucher disease patients by GBA-loaded exosomes (GBA:: EXPLOR)
A. Enzymatic activity of GBA in exosomes
the present inventors conducted an experiment of β -glucocerebrosidase activity to investigate the glucocerebrosidase degrading activity of GBA in exosomes.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Cre-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T, and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifuging (1500 × g, 30 min) a mixture of the above supernatant and ExoQuick-TC to obtain suspended exosomes.
In addition, an exosome-producing cell HEK293T which stably expresses a CIBN-EGFP-CD9 gene and a CRE-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. Then, the liquid in the exosome concentrate was removed by centrifugation (10000-14000 g, 5 min) on Amicon Ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min). Exosomes were cleaved and proteins analyzed using MPER (mammalian protein extraction reagent).
increased β -glucocerebrosidase activity of EXPLOR was observed in GBA-loaded GBA:: EXPLOR compared to mChery-loaded exosomes, confirming the loading of active GBA onto exosomes (FIG. 29).
enzymatic activity of beta-Glucocerebrosidase (GBA) on cells derived from gaucher disease patients
the inventors carried out the following experiments to confirm the recovery of the enzymatic activity of β -glucocerebrosidase on cells from gaucher's disease when treated with GBA:EXPLOR.
In 60mm petri dishes at 2X 105Density culture of individual cells derived from fibroblasts of patients with gaucher disease. Then, use mCherry:: EXPLOR (2X 10)9Exosomes) or GBA EXPLOR (1.2X 10)10exosomes) the hydrolytic activity of GBA-mCh-CRY2 was determined by detecting fluorescence using the substrate 4-methylumbelliferyl- β -D-glucopyranoside (4-MUG; Sigma), enzyme reaction was performed in 0.15% (v/v) Triton X-100(Sigma), 0.8% (w/v) sodium taurocholate (Sigma) and 10mM 4-MUG in 0.2ml citrate phosphate buffer (pH 0.5) containing 50 μ l cell lysate, after incubation for 1 hour at 37 ℃, enzyme activity was stopped using 100ul of 0.1M glycine and 0.1M NaOH (pH 10.3), the enzyme reaction product 4-methylumbelliferone (4-MU) was measured under 365nm excitation, 460nm emission conditions.
As a result, GBA-loaded GBA that the EXPLOR treatment of beta-glucosidase activity from cells of gaucher disease patients was restored (FIG. 30).
Experimental example 9: mitogen-activated kinase: p38MAP kinase
The inventors confirmed the binding of CIBN to Cry2 in cells expressing CIBN-EGFP-CD9 and p38MAP kinase-mCherry-Cry 2 under blue light at 488nm wavelength and verified the loading of p38MAP kinase in exosomes.
In order to produce p38MAP kinase-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the p38MAP kinase-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by the Tangential Flow Filtration (TFF) method.
Functional analysis of p38MAP kinase loaded exosomes in target cells:
target cells were treated with p38MAP kinase loaded exosomes to show functional activity.
P38MAP kinase loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 10: inhibitor kappa B kinase (IKK)
Binding of CIBN to CRY2 and loading of IKK in exosomes in cells expressing CIBN-EGFP-CD9 and IKK-mCherry-Cry2 at blue light wavelength 488nm was evaluated.
In order to produce IKK-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the IKK-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of IKK-loaded exosomes in target cells:
target cells were treated with IKK-loaded exosomes to show functional activity.
IKK-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 11: PTEN phosphatase
The present inventors demonstrated the binding of CIBN to CRY2 and the loading of PTEN in exosomes in cells expressing CIBN-EGFP-CD9 and PTEN-Cry2 under blue light at 488nm wavelength.
In order to produce PTEN-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the PTEN-CRY2 gene were established (FIG. 31), and purified exosomes were isolated from culture supernatants by Tangential Flow Filtration (TFF) method.
Functional analysis of PTEN-loaded exosomes in target cells:
target cells were treated with PTEN-loaded exosomes to show functional activity.
PTEN-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 12: janus kinase (JNK)
The binding of CIBN to CRY2 and the loading of JNK in exosomes was evaluated in cells expressing CIBN-EGFP-CD9 and JNK-mCherry-Cry2 under blue light at 488nm wavelength.
In order to produce JNK-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and JNK-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by Tangential Flow Filtration (TFF) method.
Functional analysis of JNK-loaded exosomes in target cells:
target cells were treated with JNK-loaded exosomes to show functional activity.
JNK-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 13: ubiquitin ligases
Binding of CIBN to Cry2 and loading of ubiquitin ligase in exosomes in cells expressing CIBN-EGFP-CD9 and ubiquitin ligase-mCherry-Cry 2 at 488nm wavelength blue light was evaluated.
In order to produce ubiquitin ligase-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the ubiquitin ligase-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of ubiquitin ligase-loaded exosomes in target cells:
target cells were treated with ubiquitin ligase-loaded exosomes to display functional activity.
Ubiquitin ligase-loaded exosomes were administered intraperitoneally or intravenously to animal models to demonstrate therapeutic efficacy.
Experimental example 14: luciferase enzyme
The present inventors confirmed the binding of CIBN to Cry2 and the loading of luciferase in exosomes in cells expressing CIBN-EGFP-CD9 and luciferase-mCherry-Cry 2 under blue light at 488nm wavelength (fig. 32).
In order to produce luciferase-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the luciferase-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by the Tangential Flow Filtration (TFF) method.
The quantified luciferase activity was analyzed based on the number of luciferase molecules (fig. 33).
Experimental example 15: peroxide oxidoreductases
< 15-1 > production of exosomes loaded with Prx I or Prx II (Prx I/II: EXPLOR)
A. Demonstration of Prx I/Prx II in exosomes
The present inventors investigated the binding of CIBN-EGFP-CD9 and PrxI/II-mCherry-CRY2 expressing CIBN and CRY2 to confirm that the amino acid sequence recorded in SEQ ID NO: prx I or Prx II loading in 7 or 8.
Specifically, after 24 hours of culture in the absence of light using a transfected pcDNA3.1(+) vector containing the CIBN-EGFP-CD9 gene and the PrxI/II-mCherry-CRY2 gene, the exosome-producing cell HEK293T was additionally cultured in Dulbecco's Modified Eagle's Medium (DMEM) without Fetal Bovine Serum (FBS) for 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before and after blue light irradiation at a wavelength of 488nm was investigated by confocal microscopy. This experiment was performed more than five times. Thus, aggregation of Prx I/II protein under blue light stimulation was confirmed (FIG. 34). Thus, the Prx I/II protein was loaded into exosomes by confirming the co-localization (yellow) of Prx I/II-mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green).
B. Generating Prx I/II:::::::::::::::::::::::::::
the present inventors performed the following experiments to obtain Prx I/II loaded exosomes.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Prx I/II-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
< 15-2 > demonstration of Prx I/II the inhibitory Effect of EXPLOR on oxidative stress-induced cytotoxicity
The present inventors carried out the following experiments to confirm the inhibitory effect of EXPLOR on oxidative stress-induced cytotoxicity.
Specifically, after changing the serum-free medium of HeLa cells, PrxI/II:: EXPLOR was treated with 100. mu.g/mL and cultured for 18 hours. By H2O2(0, 0.5, 1mM) andthe culture was continued for 8 hours. The WST assay was used to analyze cell viability.
Oxidative stress-induced cytotoxicity was significantly inhibited due to Prx I/II:: EXPLOR pretreatment (FIG. 35).
Experimental example 16: NF-kB
The binding of CIBN to CRY2 and loading of NF-. kappa.B in exosomes was evaluated in cells expressing CIBN-EGFP-CD9 and NF-. kappa.B-mCherry-Cry 2 under blue light at 488nm wavelength.
In order to produce NF-kB-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the NF-kB-mCherry-CRY 2 gene were established, and purified exosomes were separated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of NF-. kappa.B-loaded exosomes in target cells:
target cells were treated with NF-. kappa.B-loaded exosomes to show functional activity.
NF- κ B-loaded exosomes are administered to animal models intraperitoneally or intravenously to demonstrate therapeutic effect.
Experimental example 17: MyoD
The inventors demonstrated the binding of CIBN to CRY2 in cells expressing CIBN-EGFP-CD9 and MyoD-mCherry-Cry2 under blue light at 488nm wavelength (FIG. 36) and verified the loading of MyoD in exosomes.
In order to produce a large amount of MyoD-loaded exosomes, cells stably expressing the CIBN-EGFP-CD9 gene and MyoD-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Target cells were treated with MyoD-loaded exosomes to show functional activity (figure 37).
Experimental example 18: tbx18(T-box transcription factor 18)
Binding of CIBN to Cry2 and loading of Tbx18 in exosomes was evaluated at 488nm wavelength blue light in cells expressing CIBN-EGFP-CD9 and Tbx18-mCherry-Cry 2.
In order to produce a large amount of Tbx 18-loaded exosomes, cells stably expressing the CIBN-EGFP-CD9 gene and the Tbx18-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of Tbx 18-loaded exosomes in target cells:
target cells were treated with Tbx 18-loaded exosomes to show functional activity.
Tbx 18-loaded exosomes were administered intraperitoneally or intravenously to animal models to demonstrate therapeutic effect.
Experimental example 19: p53
The inventors demonstrated the binding of CIBN to Cry2 (fig. 38) and loading of PTEN in exosomes (fig. 39) in cells expressing CIBN-EGFP-CD9 and p53-mCherry-Cry2 under blue light at 488nm wavelength.
In order to produce p 53-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the p53-mCherry-CRY2 gene were established and purified exosomes were isolated from the culture supernatant by Tangential Flow Filtration (TFF) method.
Target cells were treated with p 53-loaded exosomes to show transcriptional activity (fig. 40).
P 53-loaded exosomes were administered intraperitoneally or intravenously to animal models to demonstrate therapeutic effect.
Experimental example 20: HMGB1
The present inventors demonstrated the binding of CIBN to Cry2 and the loading of HMGB1 in exosomes in cells expressing CIBN-EGFP-CD9 and HMGB1-mCherry-Cry2 under blue light at 488nm wavelength (fig. 41).
In order to produce large amount of exosomes loaded with HMGB1, cells stably expressing the CIBN-EGFP-CD9 gene and the HMGB1-mCherry-CRY2 gene were established, and purified exosomes were separated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of HMGB1 loaded exosomes in target cells:
target cells were treated with HMGB 1-loaded exosomes to show functional activity.
The HMGB 1-loaded exosomes were administered intraperitoneally or intravenously to animal models to show therapeutic effects.
Experimental example 21: supersuppressor IkappaB
< 21-1 > production of exosomes loaded with the supersuppressor IkB (supersuppressor IkB: EXPLOR)
A. Demonstration of the supersuppressor IkB in exosomes
The inventors investigated the binding of CIBN-EGFP-CD9 and the supersuppressor Iκ B-mCherry-CRY2 expressing CIBN and CRY2 to confirm that the amino acid sequence recorded in SEQ ID NO: loading of the supersuppressor IkB in 5.
Specifically, after culturing the exosome-producing cell HEK293T24 hours with pcdna3.1(+) transfected vector containing the CIBN-EGFP-CD9 gene and the hyper-repressor ikb-mCherry-CRY 2 gene in the absence of light, the exosome-producing cell HEK293T was additionally cultured in Dulbecco's Modified Eagle Medium (DMEM) without Fetal Bovine Serum (FBS) for 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before and after blue light irradiation at a wavelength of 488nm was investigated by confocal microscopy. This experiment was performed more than five times. Thus, aggregation of the superseprder I κ B protein under blue light stimulation was confirmed (fig. 42). Thus, the superseprder IkB protein was loaded into exosomes by confirming the co-localization (yellow) of the superseprder IkB-mCherry-CRY 2 (red) and CIBN-EGFP-CD9 (green).
B. Production of the supersuppressor IkB::::::::::::::::::::::::::
the present inventors performed the following experiments to obtain exosomes loaded with the supersuppressor ikb.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the supersuppressor IkappaB-mCherry-CRY 2 gene was transfected into exosome-producing cells HEK293T and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
In addition, an exosome-producing cell HEK293T which stably expresses a CIBN-EGFP-CD9 gene and a CRE-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. Then, the liquid in the exosome concentrate was removed by centrifugation (10000-14000 g, 5 min) on Amicon Ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min) with a buffer that was preferred according to experimental purpose.
< 21-2 > demonstration of the inhibitory Effect of the supersuppressor IkB ExPLOR on TNF-. alpha.mediated NF-. kappa.B Activity
the inventors performed the following experiments to demonstrate the TNF-. alpha.mediated anti-inflammatory effects using the superseprder IkB: EXPLOR.
specifically, HeLa was cultured for 3 hours in 100mg/mL mCherry: EXPLOR or hyper-repressor-ikb-mCherry: EXPLOR treated media then treated with TNF- α (10ng/mL) and incubated for an additional 30 minutes after fixation with 4% paraformaldehyde NF- κ Bp65 was stained with Alexa Fluor 488 conjugated antibody and examined using confocal microscopy in order to measure the binding activity of p65/c-Rel (NF-kB) nuclear lysates were used in the TransAM NF-kB and AP-1 assay kit (ActiveMotif, Carlsbad, CA, USA) according to the manufacturer's instructions the data presented as mean ± SEM (n ═ 3) and were applied using Tukey's post hoc test (Tukey's post test) and a significant group was determined by ANOVA test (p < 0.01).
TNF-. alpha.activated NF-. kappa.B was transported to the nucleus and NF-. kappa.BDNA binding was inhibited by pretreatment of HeLa with the supersuppressor IkB: EXPLOR (FIG. 43).
< 21-3 > demonstration of the anti-inflammatory Effect of the supersuppressor IkB: EXPLOR on animal models of collagen-induced arthritis
The inventors performed the following experiments to demonstrate the anti-inflammatory effect of the super repressor IkB EXPLOR on a mouse model of collagen-induced arthritis.
In particular, the mainly used rheumatoid arthritis model, the collagen-induced arthritis mouse model, was developed by injecting bovine type ii collagen and adjuvant into the caudal subcutaneous tissue of DBA/1 for immunization. Two collagen-induced arthritis mouse models were injected retrobulbally 4 times every 2 days with the supersepressor IkB: EXPLOR. Progression of rheumatoid arthritis symptoms was determined according to the clinical scores listed in table 3. The mean clinical score is the mean of the clinical scores from the mouse feet according to the above table.
When the hyper-repressor IkB: EXPLOR was injected retrobulbarly into a collagen-induced arthritis mouse model, rheumatoid arthritis mice showed a reduction in symptoms (FIG. 44).
[ Table 3]
| Severity scoring | Phenotypic markers |
| 0 | There were no signs of erythema and swelling |
| 1 | Erythema and mild swelling localized to the tarsal bones or ankle joints |
| 2 | Erythema and mild swelling extending from the ankle to the tarsal bones |
| 3 | Erythema and moderate swelling extending from the ankle to the metatarsal joints |
| 4 | Erythema and severe swelling covering the ankle, foot and toes, or rigidity of the limbs |
Effect of < 21-4 > srIkB-loaded exosomes on LPS-induced sepsis model
In addition, septic mice showed significantly increased survival when the supersuppressor IkB: EXPLOR was injected intraperitoneally into LPS-induced septic mouse models (FIG. 45).
Experimental example 22: pySTAT3 intrabodies
The present inventors demonstrated the binding of CIBN to CRY2 and the loading of pySTAT3 in exosomes under blue light at 488nm wavelength in cells expressing CIBN-EGFP-CD9 and pySTAT3-mCherry-Cry2 (FIG. 46).
In order to produce large amounts of exosomes loaded with pySTAT3, cells stably expressing the CIBN-EGFP-CD9 gene and pySTAT3-mCherry-CRY2 gene were established and purified exosomes were isolated from the culture supernatant by Tangential Flow Filtration (TFF) method.
Target cells were treated with pySTAT 3-loaded exosomes to assess functional activity.
The exosomes loaded with pySTAT3 were administered intraperitoneally or intravenously to animal models to demonstrate therapeutic effect.
Experimental example 23: bcl-2 related X proteins
< 23-1 > production of Bax-loaded exosomes (Bax:: EXPLOR)
A. Demonstration of Bax in exosomes
The present inventors investigated the binding of CIBN-EGFP-CD9 and Bax-mCherry-CRY2 expressing CIBN and CRY2d to confirm that the amino acid sequence recorded in amino acid SEQ ID NO: bax load in 6.
Specifically, after culturing an exosome-producing cell HEK293T24 having a pcdna3.1(+) vector comprising the CIBN-EGFP-CD9 gene and the Bax-mCherry-CRY2 gene in the absence of light for 24 hours, the exosome-producing cell HEK293T was additionally cultured in Dulbecco's Modified Eagle Medium (DMEM) without Fetal Bovine Serum (FBS) for 48 hours. After the end of the incubation, the position of the red fluorescence from mCherry before and after blue light irradiation at a wavelength of 488nm was investigated by confocal microscopy. This experiment was performed more than five times. This confirmed aggregation of Bax protein under blue light stimulation (fig. 48). Therefore, Bax protein was loaded into exosomes by confirming the binding of Bax-mCherry-CRY2 (red) and CIBN-EGFP-CD9 (green).
B. Producing Bax:: EXPLOR
The present inventors performed the following experiments to obtain Bax-loaded exosomes.
Specifically, a vector comprising the CIBN-EGRP-CD9 gene and the Bax-mCherry-CRY2 gene was transfected into an exosome-producing cell HEK293T, and these cells were cultured for 24 hours. After 24 hours of incubation, the cell culture medium was changed to be devoid of Fetal Bovine Serum (FBS) and incubated for an additional 48 hours under blue light at 488nm wavelength 50. mu.W power. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To the supernatant was added 5 volumes of ExoQuick-TC exosome precipitation solution (System Biosciences, Mountain View, California, USA) and mixed at4 ℃ for 18 hours. The exosome pellet was suspended by centrifugation (1500 × g, 30 min) of the mixture of supernatant and ExoQuick-TC to obtain suspended exosomes (fig. 8).
In addition, an exosome-producing cell HEK293T stably expressing the CIBN-EGFP-CD9 gene and the Bax-mCherry-CRY2 gene is cultured in a culture medium without fetal calf serum for 48-72 hours under blue light with the wavelength of 488nm and the power of 50 muW. After the end of the culture, a supernatant from which cell debris was removed was produced from the separated medium by centrifugation (2000 Xg, 15 minutes). To remove particles larger than 200nm from the supernatant, filtration was performed with 0.2. mu.l PES membrane (Corning). Particles smaller than 20nm were removed in the same supernatant using Tangential Flow Filtration (TFF) method, and the exosomes were concentrated and refined from the filtrate. Vivaflow 50-100kDa PES membrane (Sartorius) was used in TFF. And (3) concentrating and refining the exosome by rotating the filtrate under 1.5-2 air pressures of TFF. Then, the liquid in the exosome concentrate was removed by centrifugation (10000-14000 g, 5 min) on Amicon Ultra-0.5(100kDa) (Millipore) filters. Finally, exosomes were obtained by reverse centrifugation (10000-14000 g, 5 min) with a buffer that was preferred according to experimental purpose.
< 23-2 > confirmation of apoptosis by Bax:: EXPLOR
Bax is an apoptosis regulator, and thus Bax overexpression releases cytochrome c by binding to the mitochondrial membrane and induces apoptosis. The inventors confirmed that cytochrome c is secreted using Bax: EXPLOR.
In particular, HeLa was cultured for 12 hours in 0.1mg/mL mCherry: EXPLOR or in a medium comprising Bax-mCherry: EXPLOR. After fixation with 4% paraformaldehyde, to measure the secretion of cytochrome c, HeLa was stained with Alexa Fluor 647-conjugated antibody and imaged using a confocal microscope, and the ratio of cytochrome c was analyzed by counting the number of cells (scale bar 20 μm). Data are presented as mean ± SEM (n ═ 3) and applied using Tukey's post hoc testing, and significant groups were determined by ANOVA testing (×, p < 0.01).
As a result, a greater amount of cytochrome c release was observed in Bax: EXPLOR treated HeLa than in mCherry: EXPLOR treated HeLa (FIG. 49).
Experimental example 24: Bcl-xL
The binding of CIBN to CRY2 and the loading of Bcl-xL in exosomes was evaluated at blue light wavelength 488nm in cells expressing CIBN-EGFP-CD9 and Bcl-xL-mCherry-Cry 2.
In order to produce large amounts of Bcl-xL-loaded exosomes, cells stably expressing the CIBN-EGFP-CD9 gene and the Bcl-xL-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Functional analysis of BcL-xL loaded exosomes in target cells:
target cells were treated with Bcl-xL-loaded exosomes to show functional activity.
The Bcl-xL loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic efficacy.
Experimental example 25: AIMP
The inventors demonstrated the binding of CIBN to CRY2 in cells expressing CIBN-EGFP-CD9 and AIMP-mCherry-Cry2 under blue light at 488nm wavelength (FIG. 50) and the loading of pySTAT3 in exosomes (FIG. 51).
In order to produce large amounts of AIMP-loaded exosomes, cells stably expressing the CIBN-EGFP-CD9 gene and the AIMP-mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Target cells were treated with AIMP-loaded exosomes to assess functional activity.
The AIMP-loaded exosomes were administered to animal models intraperitoneally or intravenously to demonstrate therapeutic effects.
Experimental example 26: mcherry (fluorescent protein)
The inventors demonstrated the binding of CIBN to CRY2 in cells expressing CIBN-EGFP-CD9 and mCherry-Cry2 at blue light wavelength 488nm (FIG. 52) and the loading of AIMP in exosomes.
In order to produce mCherry-loaded exosomes in large quantities, cells stably expressing the CIBN-EGFP-CD9 gene and the mCherry-CRY2 gene were established, and purified exosomes were isolated from the culture supernatant by a Tangential Flow Filtration (TFF) method.
Experimental example 27: nucleic acid binding proteins
The binding of CIBN to Cry2 and loading of nucleic acid binding protein in exosomes was evaluated at 488nm wavelength blue light in cells expressing CIBN-EGFP-CD9 and nucleic acid binding protein-mCherry-Cry 2.
In order to produce a large amount of exosomes loaded with nucleic acid binding proteins, cells stably expressing the CIBN-EGFP-CD9 gene and the nucleic acid binding protein-mCherry-CRY 2 gene were established, and purified exosomes were isolated from the culture supernatant by the Tangential Flow Filtration (TFF) method.
Functional analysis of exosomes loaded with nucleic acid binding proteins in target cells:
target cells are treated with exosomes loaded with nucleic acid binding proteins to display functional activity.
The nucleic acid binding protein loaded exosomes are administered to animal models intraperitoneally or intravenously to demonstrate therapeutic effect.
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention, and that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.
<110> Seelrex Life sciences Co., Ltd (CELLEX LIFE SCIENCES, INCORPORATED)
<120> compositions comprising protein-loaded exosomes and methods of making and delivering the same
<130>2017FPO-09-017_PCT
<150>KR 10-2016-0126335
<151>2016-09-30
<150>KR 10-2016-0126921
<151>2016-09-30
<150>KR 10-2016-0126961
<151>2016-09-30
<150>KR 10-2016-0127486
<151>2016-10-04
<150>KR 10-2016-0132616
<151>2016-10-13
<150>KR 10-2017-0018637
<151>2017-02-10
<160>84
<170>PatentIn version 3.2
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Tyr Lys Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr
35 40 45
Phe Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asn Arg Ala Glu Asp
50 55 60
Phe Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln
65 70 75 80
Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly Leu
85 90 95
Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr Arg Arg Leu Ser
100 105 110
Glu Asp Tyr Gly Val Leu Lys Thr Asp Glu Gly Ile Ala Tyr Arg Gly
115 120 125
Leu Phe Ile Ile Asp Gly Lys Gly Val Leu Arg Gln Ile Thr Val Asn
130 135 140
Asp Leu Pro Val Gly Arg Ser Val Asp Glu Ala Leu Arg Leu Val Gln
145 150 155 160
Ala Phe Gln Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp
165 170 175
Lys Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu
180 185 190
Tyr Phe Ser Lys His Asn
195
<210>9
<211>343
<212>PRT
<213> bacteriophage P1
<400>9
Met Ser Asn Leu Leu Thr Val His Gln Asn Leu Pro Ala Leu Pro Val
1 5 10 15
Asp Ala Thr Ser Asp Glu Val Arg Lys Asn Leu Met Asp Met Phe Arg
20 25 30
Asp Arg Gln Ala Phe Ser Glu His Thr Trp Lys Met Leu Leu Ser Val
35 40 45
Cys Arg Ser Trp Ala Ala Trp Cys Lys Leu Asn Asn Arg Lys Trp Phe
50 55 60
Pro Ala Glu Pro Glu Asp Val Arg Asp Tyr Leu Leu Tyr Leu Gln Ala
65 70 75 80
Arg Gly Leu Ala Val Lys Thr Ile Gln Gln His Leu Gly Gln Leu Asn
85 90 95
Met Leu His Arg Arg Ser Gly Leu Pro Arg Pro Ser Asp Ser Asn Ala
100 105 110
Val Ser Leu Val Met Arg Arg Ile Arg Lys Glu Asn Val Asp Ala Gly
115 120 125
Glu Arg Ala Lys Gln Ala Leu Ala Phe Glu Arg Thr Asp Phe Asp Gln
130 135 140
Val Arg Ser Leu Met Glu Asn Ser Asp Arg Cys Gln Asp Ile Arg Asn
145 150 155 160
Leu Ala Phe Leu Gly Ile Ala Tyr Asn Thr Leu Leu Arg Ile Ala Glu
165 170 175
Ile Ala Arg Ile Arg Val Lys Asp Ile Ser Arg Thr Asp Gly Gly Arg
180 185 190
Met Leu Ile His Ile Gly Arg Thr Lys Thr Leu Val Ser Thr Ala Gly
195 200 205
Val Glu Lys Ala Leu Ser Leu Gly Val Thr Lys Leu Val Glu Arg Trp
210 215 220
Ile Ser Val Ser Gly Val Ala Asp Asp Pro Asn Asn Tyr Leu Phe Cys
225230 235 240
Arg Val Arg Lys Asn Gly Val Ala Ala Pro Ser Ala Thr Ser Gln Leu
245 250 255
Ser Thr Arg Ala Leu Glu Gly Ile Phe Glu Ala Thr His Arg Leu Ile
260 265 270
Tyr Gly Ala Lys Asp Asp Ser Gly Gln Arg Tyr Leu Ala Trp Ser Gly
275 280 285
His Ser Ala Arg Val Gly Ala Ala Arg Asp Met Ala Arg Ala Gly Val
290 295 300
Ser Ile Pro Glu Ile Met Gln Ala Gly Gly Trp Thr Asn Val Asn Ile
305 310 315 320
Val Met Asn Tyr Ile Arg Asn Leu Asp Ser Glu Thr Gly Ala Met Val
325 330 335
Arg Leu Leu Glu Asp Gly Asp
340
<210>10
<211>469
<212>PRT
<213> Streptococcus pyogenes (Streptococcus pyogenes)
<400>10
Met His Ser Phe Pro Pro Leu Leu Leu Leu Leu Phe Trp Gly Val Val
1 5 10 15
Ser His Ser Phe Pro Ala Thr Leu Glu Thr Gln Glu Gln Asp Val Asp
20 25 30
Leu Val Gln Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu Lys Asn Asp Gly
35 40 45
Arg Gln Val Glu Lys Arg Arg Asn Ser Gly Pro Val Val Glu Lys Leu
50 55 60
Lys Gln Met Gln Glu Phe Phe Gly Leu Lys Val Thr Gly Lys Pro Asp
65 70 75 80
Ala Glu Thr Leu Lys Val Met Lys Gln Pro Arg Cys Gly Val Pro Asp
85 90 95
Val Ala Gln Phe Val Leu Thr Glu Gly Asn Pro Arg Trp Glu Gln Thr
100 105 110
His Leu Thr Tyr Arg Ile Glu Asn Tyr Thr Pro Asp Leu Pro Arg Ala
115 120 125
Asp Val Asp His Ala Ile Glu Lys Ala Phe Gln Leu Trp Ser Asn Val
130 135 140
Thr Pro Leu Thr Phe Thr Lys Val Ser Glu Gly Gln Ala Asp Ile Met
145 150 155 160
Ile Ser Phe Val Arg Gly Asp His Arg Asp Asn Ser Pro Phe Asp Gly
165 170 175
Pro Gly Gly Asn Leu Ala His Ala Phe Gln Pro Gly Pro Gly Ile Gly
180 185 190
Gly Asp Ala His Phe Asp Glu Asp Glu Arg Trp Thr Asn Asn Phe Arg
195 200 205
Glu Tyr Asn Leu His Arg Val Ala Ala His Glu Leu Gly His Ser Leu
210 215 220
Gly Leu Ser His Ser Thr Asp Ile Gly Ala Leu Met Tyr Pro Ser Tyr
225 230 235 240
Thr Phe Ser Gly Asp Val Gln Leu Ala Gln Asp Asp Ile Asp Gly Ile
245 250 255
Gln Ala Ile Tyr Gly Arg Ser Gln Asn Pro Val Gln Pro Ile Gly Pro
260 265 270
Gln Thr Pro Lys Ala Cys Asp Ser Lys Leu Thr Phe Asp Ala Ile Thr
275 280 285
Thr Ile Arg Gly Glu Val Met Phe Phe Lys Asp Arg Phe Tyr Met Arg
290 295 300
Thr Asn Pro Phe Tyr Pro Glu Val Glu Leu Asn Phe Ile Ser Val Phe
305 310 315 320
Trp Pro Gln Leu Pro Asn Gly Leu Glu Ala Ala Tyr Glu Phe Ala Asp
325 330 335
Arg Asp Glu Val Arg Phe Phe Lys Gly Asn Lys Tyr Trp Ala Val Gln
340 345 350
Gly Gln Asn Val Leu His Gly Tyr Pro Lys Asp Ile Tyr Ser Ser Phe
355 360 365
Gly Phe Pro Arg Thr Val Lys His Ile Asp Ala Ala Leu Ser Glu Glu
370 375 380
Asn Thr Gly Lys Thr Tyr Phe Phe Val Ala Asn Lys Tyr Trp Arg Tyr
385 390 395 400
Asp Glu Tyr Lys Arg Ser Met Asp Pro Gly Tyr Pro Lys Met Ile Ala
405 410 415
His Asp Phe Pro Gly Ile GlyHis Lys Val Asp Ala Val Phe Met Lys
420 425 430
Asp Gly Phe Phe Tyr Phe Phe His Gly Thr Arg Gln Tyr Lys Phe Asp
435 440 445
Pro Lys Thr Lys Arg Ile Leu Thr Leu Gln Lys Ala Asn Ser Trp Phe
450 455 460
Asn Cys Arg Lys Asn
465
<210>11
<211>1300
<212>PRT
<213> Francisella tularensis (Francisella tularensis)
<400>11
Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr
1 5 10 15
Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys
20 25 30
Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys
35 40 45
Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu
5055 60
Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser
65 70 75 80
Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys
85 90 95
Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr
100 105 110
Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile
115 120 125
Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln
130 135 140
Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr
145 150 155 160
Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr
165 170 175
Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser
180 185 190
Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu
195 200 205
Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys
210 215 220
Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu
225 230 235 240
Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg
245 250 255
Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr
260 265 270
Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys
275 280 285
Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile
290 295 300
Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys
305 310 315 320
Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser
325 330 335
Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met
340 345 350
Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys
355 360 365
Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln
370 375 380
Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr
385 390 395 400
Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala
405 410 415
Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn
420 425 430
Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala
435 440 445
Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn
450 455 460
Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala
465 470 475 480
Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys
485 490 495
Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys
500 505 510
Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp
515 520 525
Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His
530 535 540
Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His
545 550 555 560
Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val
565 570 575
Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser
580 585 590
Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly
595 600 605
Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys
610615 620
Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile
625 630 635 640
Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys
645 650 655
Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val
660 665 670
Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile
675 680 685
Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln
690 695 700
Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe
705 710 715 720
Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp
725 730 735
Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu
740 745 750
Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn
755 760 765
Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr
770 775 780
Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg
785 790 795 800
Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn
805 810 815
Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr
820 825 830
Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala
835 840 845
Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu
850 855 860
Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe
865 870 875 880
His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe
885 890 895
Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His
900 905 910
Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu
915 920 925
Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile
930 935 940
Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile
945 950 955 960
Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn
965 970 975
Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile
980 985 990
Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu
995 1000 1005
Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val Tyr
1010 1015 1020
Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu Val Phe
1025 1030 1035 1040
Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg Ala Tyr Gln
1045 1050 1055
Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly Lys Gln Thr Gly
1060 1065 1070
Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser Lys Ile Cys Pro Val
1075 1080 1085
Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys Tyr Glu Ser Val Ser Lys
1090 1095 1100
Ser Gln Glu Phe Phe Ser Lys Phe Asp Lys Ile Cys Tyr Asn Leu Asp
1105 1110 1115 1120
Lys Gly Tyr Phe Glu Phe Ser Phe Asp Tyr Lys Asn Phe Gly Asp Lys
1125 1130 1135
Ala Ala Lys Gly Lys Trp Thr Ile Ala Ser Phe Gly Ser Arg Leu Ile
1140 1145 1150
Asn Phe Arg Asn Ser Asp Lys Asn His Asn Trp Asp Thr Arg Glu Val
1155 1160 1165
Tyr Pro Thr Lys Glu Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu
1170 1175 1180
Tyr Gly His Gly Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp
1185 1190 1195 1200
Lys Lys Phe Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln
1205 1210 1215
Met Arg Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro
1220 1225 1230
Val Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys
1235 1240 1245
Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu
1250 1255 1260
Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu Gly Lys
1265 1270 1275 1280
Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu Phe Val Gln
1285 1290 1295
Asn Arg Asn Asn
1300
<210>12
<211>536
<212>PRT
<213> human (Homo sapiens)
<400>12
Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser
1 5 10 15
Arg Val Ser Ile Met Ala Gly Ser Leu Thr Gly Leu Leu Leu Leu Gln
20 25 30
Ala Val Ser Trp Ala Ser Gly Ala Arg Pro Cys Ile Pro Lys Ser Phe
35 40 45
Gly Tyr Ser Ser Val Val Cys Val Cys Asn Ala Thr Tyr Cys Asp Ser
50 55 60
Phe Asp Pro Pro Thr Phe Pro Ala Leu Gly Thr Phe Ser Arg Tyr Glu
65 70 75 80
Ser Thr Arg Ser Gly Arg Arg Met Glu Leu Ser Met Gly Pro Ile Gln
85 90 95
Ala Asn His Thr Gly Thr Gly Leu Leu Leu Thr Leu Gln Pro Glu Gln
100 105 110
Lys Phe Gln Lys Val Lys Gly Phe Gly Gly Ala Met Thr Asp Ala Ala
115 120 125
Ala Leu Asn Ile Leu Ala Leu Ser Pro Pro Ala Gln Asn Leu Leu Leu
130 135 140
Lys Ser Tyr Phe Ser Glu Glu Gly Ile Gly Tyr Asn Ile Ile Arg Val
145 150 155 160
Pro Met Ala Ser Cys Asp Phe Ser Ile Arg Thr Tyr Thr Tyr Ala Asp
165 170 175
Thr Pro Asp Asp Phe Gln Leu His Asn Phe Ser Leu Pro Glu Glu Asp
180 185 190
Thr Lys Leu Lys Ile Pro Leu Ile His Arg Ala Leu Gln Leu Ala Gln
195 200 205
Arg Pro Val Ser Leu Leu Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu
210 215 220
Lys Thr Asn Gly Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro
225 230 235 240
Gly Asp Ile Tyr His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu
245 250 255
Asp Ala Tyr Ala Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu
260 265270
Asn Glu Pro Ser Ala Gly Leu Leu Ser Gly Tyr Pro Phe Gln Cys Leu
275 280 285
Gly Phe Thr Pro Glu His Gln Arg Asp Phe Ile Ala Arg Asp Leu Gly
290 295 300
Pro Thr Leu Ala Asn Ser Thr His His Asn Val Arg Leu Leu Met Leu
305 310 315 320
Asp Asp Gln Arg Leu Leu Leu Pro His Trp Ala Lys Val Val Leu Thr
325 330 335
Asp Pro Glu Ala Ala Lys Tyr Val His Gly Ile Ala Val His Trp Tyr
340 345 350
Leu Asp Phe Leu Ala Pro Ala Lys Ala Thr Leu Gly Glu Thr His Arg
355 360 365
Leu Phe Pro Asn Thr Met Leu Phe Ala Ser Glu Ala Cys Val Gly Ser
370 375 380
Lys Phe Trp Glu Gln Ser Val Arg Leu Gly Ser Trp Asp Arg Gly Met
385 390 395 400
Gln Tyr Ser His Ser Ile Ile Thr Asn Leu Leu Tyr His Val Val Gly
405 410 415
Trp Thr Asp Trp Asn Leu Ala Leu Asn Pro Glu Gly Gly Pro Asn Trp
420 425 430
Val Arg Asn Phe Val Asp Ser Pro Ile Ile Val Asp Ile Thr Lys Asp
435 440 445
Thr Phe Tyr Lys Gln Pro Met Phe Tyr His Leu Gly His Phe Ser Lys
450 455 460
Phe Ile Pro Glu Gly Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys
465 470 475 480
Asn Asp Leu Asp Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val
485 490 495
Val Val Val Leu Asn Arg Ser Ser Lys Asp Val Pro Leu Thr Ile Lys
500 505 510
Asp Pro Ala Val Gly Phe Leu Glu Thr Ile Ser Pro Gly Tyr Ser Ile
515 520 525
His Thr Tyr Leu Trp Arg Arg Gln
530 535
<210>13
<211>469
<212>PRT
<213> human (Homo sapiens)
<400>13
Met His Ser Phe Pro Pro Leu Leu Leu Leu Leu Phe Trp Gly Val Val
1 5 10 15
Ser His Ser Phe Pro Ala Thr Leu Glu Thr Gln Glu Gln Asp Val Asp
20 25 30
Leu Val Gln Lys Tyr Leu Glu Lys Tyr Tyr Asn Leu Lys Asn Asp Gly
35 40 45
Arg Gln Val Glu Lys Arg Arg Asn Ser Gly Pro Val Val Glu Lys Leu
50 55 60
Lys Gln Met Gln Glu Phe Phe Gly Leu Lys Val Thr Gly Lys Pro Asp
65 70 75 80
Ala Glu Thr Leu Lys Val Met Lys Gln Pro Arg Cys Gly Val Pro Asp
85 90 95
Val Ala Gln Phe Val Leu Thr Glu Gly Asn Pro Arg Trp Glu Gln Thr
100 105 110
His Leu Thr Tyr Arg Ile Glu Asn Tyr Thr Pro Asp Leu Pro Arg Ala
115120 125
Asp Val Asp His Ala Ile Glu Lys Ala Phe Gln Leu Trp Ser Asn Val
130 135 140
Thr Pro Leu Thr Phe Thr Lys Val Ser Glu Gly Gln Ala Asp Ile Met
145 150 155 160
Ile Ser Phe Val Arg Gly Asp His Arg Asp Asn Ser Pro Phe Asp Gly
165 170 175
Pro Gly Gly Asn Leu Ala His Ala Phe Gln Pro Gly Pro Gly Ile Gly
180 185 190
Gly Asp Ala His Phe Asp Glu Asp Glu Arg Trp Thr Asn Asn Phe Arg
195 200 205
Glu Tyr Asn Leu His Arg Val Ala Ala His Glu Leu Gly His Ser Leu
210 215 220
Gly Leu Ser His Ser Thr Asp Ile Gly Ala Leu Met Tyr Pro Ser Tyr
225 230 235 240
Thr Phe Ser Gly Asp Val Gln Leu Ala Gln Asp Asp Ile Asp Gly Ile
245 250 255
Gln Ala Ile Tyr Gly Arg Ser Gln Asn Pro Val Gln Pro Ile Gly Pro
260 265 270
Gln Thr Pro Lys Ala Cys Asp Ser Lys Leu Thr Phe Asp Ala Ile Thr
275 280 285
Thr Ile Arg Gly Glu Val Met Phe Phe Lys Asp Arg Phe Tyr Met Arg
290 295 300
Thr Asn Pro Phe Tyr Pro Glu Val Glu Leu Asn Phe Ile Ser Val Phe
305 310 315 320
Trp Pro Gln Leu Pro Asn Gly Leu Glu Ala Ala Tyr Glu Phe Ala Asp
325 330 335
Arg Asp Glu Val Arg Phe Phe Lys Gly Asn Lys Tyr Trp Ala Val Gln
340 345 350
Gly Gln Asn Val Leu His Gly Tyr Pro Lys Asp Ile Tyr Ser Ser Phe
355 360 365
Gly Phe Pro Arg Thr Val Lys His Ile Asp Ala Ala Leu Ser Glu Glu
370 375 380
Asn Thr Gly Lys Thr Tyr Phe Phe Val Ala Asn Lys Tyr Trp Arg Tyr
385 390 395 400
Asp Glu Tyr Lys Arg SerMet Asp Pro Gly Tyr Pro Lys Met Ile Ala
405 410 415
His Asp Phe Pro Gly Ile Gly His Lys Val Asp Ala Val Phe Met Lys
420 425 430
Asp Gly Phe Phe Tyr Phe Phe His Gly Thr Arg Gln Tyr Lys Phe Asp
435 440 445
Pro Lys Thr Lys Arg Ile Leu Thr Leu Gln Lys Ala Asn Ser Trp Phe
450 455 460
Asn Cys Arg Lys Asn
465
<210>14
<211>207
<212>PRT
<213> human (Homo sapiens)
<400>14
Met Ala Pro Phe Glu Pro Leu Ala Ser Gly Ile Leu Leu Leu Leu Trp
1 5 10 15
Leu Ile Ala Pro Ser Arg Ala Cys Thr Cys Val Pro Pro His Pro Gln
20 25 30
Thr Ala Phe Cys Asn Ser Asp Leu Val Ile Arg Ala Lys Phe Val Gly
35 4045
Thr Pro Glu Val Asn Gln Thr Thr Leu Tyr Gln Arg Tyr Glu Ile Lys
50 55 60
Met Thr Lys Met Tyr Lys Gly Phe Gln Ala Leu Gly Asp Ala Ala Asp
65 70 75 80
Ile Arg Phe Val Tyr Thr Pro Ala Met Glu Ser Val Cys Gly Tyr Phe
85 90 95
His Arg Ser His Asn Arg Ser Glu Glu Phe Leu Ile Ala Gly Lys Leu
100 105 110
Gln Asp Gly Leu Leu His Ile Thr Thr Cys Ser Phe Val Ala Pro Trp
115 120 125
Asn Ser Leu Ser Leu Ala Gln Arg Arg Gly Phe Thr Lys Thr Tyr Thr
130 135 140
Val Gly Cys Glu Glu Cys Thr Val Phe Pro Cys Leu Ser Ile Pro Cys
145 150 155 160
Lys Leu Gln Ser Gly Thr His Cys Leu Trp Thr Asp Gln Leu Leu Gln
165 170 175
Gly Ser Glu Lys Gly Phe Gln Ser Arg His Leu Ala Cys Leu Pro Arg
180 185 190
Glu Pro Gly Leu Cys Thr Trp Gln Ser Leu Arg Ser Gln Ile Ala
195 200 205
<210>15
<211>220
<212>PRT
<213> human (Homo sapiens)
<400>15
Met Gly Ala Ala Ala Arg Thr Leu Arg Leu Ala Leu Gly Leu Leu Leu
1 5 10 15
Leu Ala Thr Leu Leu Arg Pro Ala Asp Ala Cys Ser Cys Ser Pro Val
20 25 30
His Pro Gln Gln Ala Phe Cys Asn Ala Asp Val Val Ile Arg Ala Lys
35 40 45
Ala Val Ser Glu Lys Glu Val Asp Ser Gly Asn Asp Ile Tyr Gly Asn
50 55 60
Pro Ile Lys Arg Ile Gln Tyr Glu Ile Lys Gln Ile Lys Met Phe Lys
65 70 75 80
Gly Pro Glu Lys Asp Ile Glu Phe Ile Tyr Thr Ala Pro Ser Ser Ala
8590 95
Val Cys Gly Val Ser Leu Asp Val Gly Gly Lys Lys Glu Tyr Leu Ile
100 105 110
Ala Gly Lys Ala Glu Gly Asp Gly Lys Met His Ile Thr Leu Cys Asp
115 120 125
Phe Ile Val Pro Trp Asp Thr Leu Ser Thr Thr Gln Lys Lys Ser Leu
130 135 140
Asn His Arg Tyr Gln Met Gly Cys Glu Cys Lys Ile Thr Arg Cys Pro
145 150 155 160
Met Ile Pro Cys Tyr Ile Ser Ser Pro Asp Glu Cys Leu Trp Met Asp
165 170 175
Trp Val Thr Glu Lys Asn Ile Asn Gly His Gln Ala Lys Phe Phe Ala
180 185 190
Cys Ile Lys Arg Ser Asp Gly Ser Cys Ala Trp Tyr Arg Gly Ala Ala
195 200 205
Pro Pro Lys Gln Glu Phe Leu Asp Ile Glu Asp Pro
210 215 220
<210>16
<211>211
<212>PRT
<213> human (Homo sapiens)
<400>16
Met Thr Pro Trp Leu Gly Leu Ile Val Leu Leu Gly Ser Trp Ser Leu
1 5 10 15
Gly Asp Trp Gly Ala Glu Ala Cys Thr Cys Ser Pro Ser His Pro Gln
20 25 30
Asp Ala Phe Cys Asn Ser Asp Ile Val Ile Arg Ala Lys Val Val Gly
35 40 45
Lys Lys Leu Val Lys Glu Gly Pro Phe Gly Thr Leu Val Tyr Thr Ile
50 55 60
Lys Gln Met Lys Met Tyr Arg Gly Phe Thr Lys Met Pro His Val Gln
65 70 75 80
Tyr Ile His Thr Glu Ala Ser Glu Ser Leu Cys Gly Leu Lys Leu Glu
85 90 95
Val Asn Lys Tyr Gln Tyr Leu Leu Thr Gly Arg Val Tyr Asp Gly Lys
100 105 110
Met Tyr Thr Gly Leu Cys Asn Phe Val Glu Arg Trp Asp Gln Leu Thr
115120 125
Leu Ser Gln Arg Lys Gly Leu Asn Tyr Arg Tyr His Leu Gly Cys Asn
130 135 140
Cys Lys Ile Lys Ser Cys Tyr Tyr Leu Pro Cys Phe Val Thr Ser Lys
145 150 155 160
Asn Glu Cys Leu Trp Thr Asp Met Leu Ser Asn Phe Gly Tyr Pro Gly
165 170 175
Tyr Gln Ser Lys His Tyr Ala Cys Ile Arg Gln Lys Gly Gly Tyr Cys
180 185 190
Ser Trp Tyr Arg Gly Trp Ala Pro Pro Asp Lys Ser Ile Ile Asn Ala
195 200 205
Thr Asp Pro
210
<210>17
<211>224
<212>PRT
<213> human (Homo sapiens)
<400>17
Met Pro Gly Ser Pro Arg Pro Ala Pro Ser Trp Val Leu Leu Leu Arg
1 5 10 15
Leu Leu Ala Leu Leu Arg Pro Pro GlyLeu Gly Glu Ala Cys Ser Cys
20 25 30
Ala Pro Ala His Pro Gln Gln His Ile Cys His Ser Ala Leu Val Ile
35 40 45
Arg Ala Lys Ile Ser Ser Glu Lys Val Val Pro Ala Ser Ala Asp Pro
50 55 60
Ala Asp Thr Glu Lys Met Leu Arg Tyr Glu Ile Lys Gln Ile Lys Met
65 70 75 80
Phe Lys Gly Phe Glu Lys Val Lys Asp Val Gln Tyr Ile Tyr Thr Pro
85 90 95
Phe Asp Ser Ser Leu Cys Gly Val Lys Leu Glu Ala Asn Ser Gln Lys
100 105 110
Gln Tyr Leu Leu Thr Gly Gln Val Leu Ser Asp Gly Lys Val Phe Ile
115 120 125
His Leu Cys Asn Tyr Ile Glu Pro Trp Glu Asp Leu Ser Leu Val Gln
130 135 140
Arg Glu Ser Leu Asn His His Tyr His Leu Asn Cys Gly Cys Gln Ile
145 150 155 160
Thr Thr Cys Tyr Thr Val Pro Cys Thr Ile Ser Ala Pro Asn Glu Cys
165 170 175
Leu Trp Thr Asp Trp Leu Leu Glu Arg Lys Leu Tyr Gly Tyr Gln Ala
180 185 190
Gln His Tyr Val Cys Met Lys His Val Asp Gly Thr Cys Ser Trp Tyr
195 200 205
Arg Gly His Leu Pro Leu Arg Lys Glu Phe Val Asp Ile Val Gln Pro
210 215 220
<210>18
<211>404
<212>PRT
<213> human (Homo sapiens)
<400>18
Met Ala Asp Lys Val Leu Lys Glu Lys Arg Lys Leu Phe Ile Arg Ser
1 5 10 15
Met Gly Glu Gly Thr Ile Asn Gly Leu Leu Asp Glu Leu Leu Gln Thr
20 25 30
Arg Val Leu Asn Lys Glu Glu Met Glu Lys Val Lys Arg Glu Asn Ala
35 40 45
Thr Val Met Asp Lys Thr Arg Ala Leu Ile Asp Ser Val Ile Pro Lys
50 55 60
Gly Ala Gln Ala Cys Gln Ile Cys Ile Thr Tyr Ile Cys Glu Glu Asp
65 70 75 80
Ser Tyr Leu Ala Gly Thr Leu Gly Leu Ser Ala Asp Gln Thr Ser Gly
85 90 95
Asn Tyr Leu Asn Met Gln Asp Ser Gln Gly Val Leu Ser Ser Phe Pro
100 105 110
Ala Pro Gln Ala Val Gln Asp Asn Pro Ala Met Pro Thr Ser Ser Gly
115 120 125
Ser Glu Gly Asn Val Lys Leu Cys Ser Leu Glu Glu Ala Gln Arg Ile
130 135 140
Trp Lys Gln Lys Ser Ala Glu Ile Tyr Pro Ile Met Asp Lys Ser Ser
145 150 155 160
Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Glu Glu Phe Asp Ser Ile
165 170 175
Pro Arg Arg Thr Gly Ala Glu Val Asp Ile Thr Gly Met Thr Met Leu
180 185 190
Leu Gln Asn Leu Gly Tyr Ser Val Asp Val Lys Lys Asn Leu Thr Ala
195 200 205
Ser Asp Met Thr Thr Glu Leu Glu Ala Phe Ala His Arg Pro Glu His
210 215 220
Lys Thr Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Gly Ile Arg
225 230 235 240
Glu Gly Ile Cys Gly Lys Lys His Ser Glu Gln Val Pro Asp Ile Leu
245 250 255
Gln Leu Asn Ala Ile Phe Asn Met Leu Asn Thr Lys Asn Cys Pro Ser
260 265 270
Leu Lys Asp Lys Pro Lys Val Ile Ile Ile Gln Ala Cys Arg Gly Asp
275 280 285
Ser Pro Gly Val Val Trp Phe Lys Asp Ser Val Gly Val Ser Gly Asn
290 295 300
Leu Ser Leu Pro Thr Thr Glu Glu Phe Glu Asp Asp Ala Ile Lys Lys
305 310 315 320
Ala His Ile Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro Asp
325330 335
Asn Val Ser Trp Arg His Pro Thr Met Gly Ser Val Phe Ile Gly Arg
340 345 350
Leu Ile Glu His Met Gln Glu Tyr Ala Cys Ser Cys Asp Val Glu Glu
355 360 365
Ile Phe Arg Lys Val Arg Phe Ser Phe Glu Gln Pro Asp Gly Arg Ala
370 375 380
Gln Met Pro Thr Thr Glu Arg Val Thr Leu Thr Arg Cys Phe Tyr Leu
385 390 395 400
Phe Pro Gly His
<210>19
<211>452
<212>PRT
<213> human (Homo sapiens)
<400>19
Met Ala Ala Pro Ser Ala Gly Ser Trp Ser Thr Phe Gln His Lys Glu
1 5 10 15
Leu Met Ala Ala Asp Arg Gly Arg Arg Ile Leu Gly Val Cys Gly Met
20 25 30
His Pro His His Gln Glu Thr Leu Lys Lys Asn Arg Val Val Leu Ala
35 40 45
Lys Gln Leu Leu Leu Ser Glu Leu Leu Glu His Leu Leu Glu Lys Asp
50 55 60
Ile Ile Thr Leu Glu Met Arg Glu Leu Ile Gln Ala Lys Val Gly Ser
65 70 75 80
Phe Ser Gln Asn Val Glu Leu Leu Asn Leu Leu Pro Lys Arg Gly Pro
85 90 95
Gln Ala Phe Asp Ala Phe Cys Glu Ala Leu Arg Glu Thr Lys Gln Gly
100 105 110
His Leu Glu Asp Met Leu Leu Thr Thr Leu Ser Gly Leu Gln His Val
115 120 125
Leu Pro Pro Leu Ser Cys Asp Tyr Asp Leu Ser Leu Pro Phe Pro Val
130 135 140
Cys Glu Ser Cys Pro Leu Tyr Lys Lys Leu Arg Leu Ser Thr Asp Thr
145 150 155 160
Val Glu His Ser Leu Asp Asn Lys Asp Gly Pro Val Cys Leu Gln Val
165 170 175
Lys Pro Cys Thr Pro Glu Phe Tyr Gln Thr His Phe Gln Leu Ala Tyr
180 185 190
Arg Leu Gln Ser Arg Pro Arg Gly Leu Ala Leu Val Leu Ser Asn Val
195 200 205
His Phe Thr Gly Glu Lys Glu Leu Glu Phe Arg Ser Gly Gly Asp Val
210 215 220
Asp His Ser Thr Leu Val Thr Leu Phe Lys Leu Leu Gly Tyr Asp Val
225 230 235 240
His Val Leu Cys Asp Gln Thr Ala Gln Glu Met Gln Glu Lys Leu Gln
245 250 255
Asn Phe Ala Gln Leu Pro Ala His Arg Val Thr Asp Ser Cys Ile Val
260 265 270
Ala Leu Leu Ser His Gly Val Glu Gly Ala Ile Tyr Gly Val Asp Gly
275 280 285
Lys Leu Leu Gln Leu Gln Glu Val Phe Gln Leu Phe Asp Asn Ala Asn
290 295 300
Cys Pro Ser Leu Gln Asn Lys Pro Lys Met Phe Phe Ile Gln Ala Cys
305 310 315 320
Arg Gly Asp Glu Thr Asp Arg Gly Val Asp Gln Gln Asp Gly Lys Asn
325 330 335
His Ala Gly Ser Pro Gly Cys Glu Glu Ser Asp Ala Gly Lys Glu Lys
340 345 350
Leu Pro Lys Met Arg Leu Pro Thr Arg Ser Asp Met Ile Cys Gly Tyr
355 360 365
Ala Cys Leu Lys Gly Thr Ala Ala Met Arg Asn Thr Lys Arg Gly Ser
370 375 380
Trp Tyr Ile Glu Ala Leu Ala Gln Val Phe Ser Glu Arg Ala Cys Asp
385 390 395 400
Met His Val Ala Asp Met Leu Val Lys Val Asn Ala Leu Ile Lys Asp
405 410 415
Arg Glu Gly Tyr Ala Pro Gly Thr Glu Phe His Arg Cys Lys Glu Met
420 425 430
Ser Glu Tyr Cys Ser Thr Leu Cys Arg His Leu Tyr Leu Phe Pro Gly
435 440 445
His Pro Pro Thr
450
<210>20
<211>277
<212>PRT
<213> human (Homo sapiens)
<400>20
Met Glu Asn Thr Glu Asn Ser Val Asp Ser Lys Ser Ile Lys Asn Leu
1 5 10 15
Glu Pro Lys Ile Ile His Gly Ser Glu Ser Met Asp Ser Gly Ile Ser
20 25 30
Leu Asp Asn Ser Tyr Lys Met Asp Tyr Pro Glu Met Gly Leu Cys Ile
35 40 45
Ile Ile Asn Asn Lys Asn Phe His Lys Ser Thr Gly Met Thr Ser Arg
50 55 60
Ser Gly Thr Asp Val Asp Ala Ala Asn Leu Arg Glu Thr Phe Arg Asn
65 70 75 80
Leu Lys Tyr Glu Val Arg Asn Lys Asn Asp Leu Thr Arg Glu Glu Ile
85 90 95
Val Glu Leu Met Arg Asp Val Ser Lys Glu Asp His Ser Lys Arg Ser
100 105 110
Ser Phe Val Cys Val Leu Leu Ser His Gly Glu GluGly Ile Ile Phe
115 120 125
Gly Thr Asn Gly Pro Val Asp Leu Lys Lys Ile Thr Asn Phe Phe Arg
130 135 140
Gly Asp Arg Cys Arg Ser Leu Thr Gly Lys Pro Lys Leu Phe Ile Ile
145 150 155 160
Gln Ala Cys Arg Gly Thr Glu Leu Asp Cys Gly Ile Glu Thr Asp Ser
165 170 175
Gly Val Asp Asp Asp Met Ala Cys His Lys Ile Pro Val Glu Ala Asp
180 185 190
Phe Leu Tyr Ala Tyr Ser Thr Ala Pro Gly Tyr Tyr Ser Trp Arg Asn
195 200 205
Ser Lys Asp Gly Ser Trp Phe Ile Gln Ser Leu Cys Ala Met Leu Lys
210 215 220
Gln Tyr Ala Asp Lys Leu Glu Phe Met His Ile Leu Thr Arg Val Asn
225 230 235 240
Arg Lys Val Ala Thr Glu Phe Glu Ser Phe Ser Phe Asp Ala Thr Phe
245 250 255
His Ala Lys Lys Gln Ile Pro Cys Ile Val Ser Met Leu Thr Lys Glu
260 265 270
Leu Tyr Phe Tyr His
275
<210>21
<211>377
<212>PRT
<213> human (Homo sapiens)
<400>21
Met Ala Glu Gly Asn His Arg Lys Lys Pro Leu Lys Val Leu Glu Ser
1 5 10 15
Leu Gly Lys Asp Phe Leu Thr Gly Val Leu Asp Asn Leu Val Glu Gln
20 25 30
Asn Val Leu Asn Trp Lys Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Ala
35 40 45
Lys Thr Glu Asp Lys Val Arg Val Met Ala Asp Ser Met Gln Glu Lys
50 55 60
Gln Arg Met Ala Gly Gln Met Leu Leu Gln Thr Phe Phe Asn Ile Asp
65 70 75 80
Gln Ile Ser Pro Asn Lys Lys Ala His Pro Asn Met Glu Ala Gly Pro
85 90 95
Pro Glu Ser Gly Glu Ser Thr Asp Ala Leu Lys Leu Cys Pro His Glu
100 105 110
Glu Phe Leu Arg Leu Cys Lys Glu Arg Ala Glu Glu Ile Tyr Pro Ile
115 120 125
Lys Glu Arg Asn Asn Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr
130 135 140
Glu Phe Asp His Leu Pro Pro Arg Asn Gly Ala Asp Phe Asp Ile Thr
145 150 155 160
Gly Met Lys Glu Leu Leu Glu Gly Leu Asp Tyr Ser Val Asp Val Glu
165 170 175
Glu Asn Leu Thr Ala Arg Asp Met Glu Ser Ala Leu Arg Ala Phe Ala
180 185 190
Thr Arg Pro Glu His Lys Ser Ser Asp Ser Thr Phe Leu Val Leu Met
195 200 205
Ser His Gly Ile Leu Glu Gly Ile Cys Gly Thr Val His Asp Glu Lys
210 215 220
Lys Pro Asp Val Leu Leu Tyr Asp Thr Ile Phe Gln Ile Phe Asn Asn
225 230 235 240
Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln
245 250 255
Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Arg Asp Ser Pro
260 265 270
Ala Ser Leu Glu Val Ala Ser Ser Gln Ser Ser Glu Asn Leu Glu Glu
275 280 285
Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala Phe Cys
290 295 300
Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ser Thr Met Gly Ser
305 310 315 320
Ile Phe Ile Thr Gln Leu Ile Thr Cys Phe Gln Lys Tyr Ser Trp Cys
325 330 335
Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Thr
340 345 350
Pro Arg Ala Lys Ala Gln Met Pro Thr Ile Glu Arg Leu Ser Met Thr
355 360 365
Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
370 375
<210>22
<211>434
<212>PRT
<213> human (Homo sapiens)
<400>22
Met Ala Glu Asp Ser Gly Lys Lys Lys Arg Arg Lys Asn Phe Glu Ala
1 5 10 15
Met Phe Lys Gly Ile Leu Gln Ser Gly Leu Asp Asn Phe Val Ile Asn
20 25 30
His Met Leu Lys Asn Asn Val Ala Gly Gln Thr Ser Ile Gln Thr Leu
35 40 45
Val Pro Asn Thr Asp Gln Lys Ser Thr Ser Val Lys Lys Asp Asn His
50 55 60
Lys Lys Lys Thr Val Lys Met Leu Glu Tyr Leu Gly Lys Asp Val Leu
65 70 75 80
His Gly Val Phe Asn Tyr Leu Ala Lys His Asp Val Leu Thr Leu Lys
85 90 95
Glu Glu Glu Lys Lys Lys Tyr Tyr Asp Thr Lys Ile Glu Asp Lys Ala
100 105 110
Leu Ile Leu Val Asp Ser Leu Arg Lys Asn Arg Val Ala His Gln Met
115 120 125
Phe Thr Gln Thr Leu Leu Asn Met Asp Gln Lys Ile Thr Ser Val Lys
130 135 140
Pro Leu Leu Gln Ile Glu Ala Gly Pro Pro Glu Ser Ala Glu Ser Thr
145 150 155 160
Asn Ile Leu Lys Leu Cys Pro Arg Glu Glu Phe Leu Arg Leu Cys Lys
165 170 175
Lys Asn His Asp Glu Ile Tyr Pro Ile Lys Lys Arg Glu Asp Arg Arg
180 185 190
Arg Leu Ala Leu Ile Ile Cys Asn Thr Lys Phe Asp His Leu Pro Ala
195 200 205
Arg Asn Gly Ala His Tyr Asp Ile Val Gly Met Lys Arg Leu Leu Gln
210 215 220
Gly Leu Gly Tyr Thr Val Val Asp Glu Lys Asn Leu Thr Ala Arg Asp
225 230 235 240
Met Glu SerVal Leu Arg Ala Phe Ala Ala Arg Pro Glu His Lys Ser
245 250 255
Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Ile Leu Glu Gly
260 265 270
Ile Cys Gly Thr Ala His Lys Lys Lys Lys Pro Asp Val Leu Leu Tyr
275 280 285
Asp Thr Ile Phe Gln Ile Phe Asn Asn Arg Asn Cys Leu Ser Leu Lys
290 295 300
Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly Glu Lys His
305 310 315 320
Gly Glu Leu Trp Val Arg Asp Ser Pro Ala Ser Leu Ala Leu Ile Ser
325 330 335
Ser Gln Ser Ser Glu Asn Leu Glu Ala Asp Ser Val Cys Lys Ile His
340 345 350
Glu Glu Lys Asp Phe Ile Ala Phe Cys Ser Ser Thr Pro His Asn Val
355 360 365
Ser Trp Arg Asp Arg Thr Arg Gly Ser Ile Phe Ile Thr Glu Leu Ile
370 375 380
Thr Cys Phe Gln Lys Tyr Ser Cys Cys Cys His Leu Met Glu Ile Phe
385 390 395 400
Arg Lys Val Gln Lys Ser Phe Glu Val Pro Gln Ala Lys Ala Gln Met
405 410 415
Pro Thr Ile Glu Arg Ala Thr Leu Thr Arg Asp Phe Tyr Leu Phe Pro
420 425 430
Gly Asn
<210>23
<211>293
<212>PRT
<213> human (Homo sapiens)
<400>23
Met Ser Ser Ala Ser Gly Leu Arg Arg Gly His Pro Ala Gly Gly Glu
1 5 10 15
Glu Asn Met Thr Glu Thr Asp Ala Phe Tyr Lys Arg Glu Met Phe Asp
20 25 30
Pro Ala Glu Lys Tyr Lys Met Asp His Arg Arg Arg Gly Ile Ala Leu
35 40 45
Ile Phe Asn His Glu Arg Phe Phe Trp His Leu Thr Leu Pro Glu Arg
50 5560
Arg Gly Thr Cys Ala Asp Arg Asp Asn Leu Thr Arg Arg Phe Ser Asp
65 70 75 80
Leu Gly Phe Glu Val Lys Cys Phe Asn Asp Leu Lys Ala Glu Glu Leu
85 90 95
Leu Leu Lys Ile His Glu Val Ser Thr Val Ser His Ala Asp Ala Asp
100 105 110
Cys Phe Val Cys Val Phe Leu Ser His Gly Glu Gly Asn His Ile Tyr
115 120 125
Ala Tyr Asp Ala Lys Ile Glu Ile Gln Thr Leu Thr Gly Leu Phe Lys
130 135 140
Gly Asp Lys Cys His Ser Leu Val Gly Lys Pro Lys Ile Phe Ile Ile
145 150 155 160
Gln Ala Cys Arg Gly Asn Gln His Asp Val Pro Val Ile Pro Leu Asp
165 170 175
Val Val Asp Asn Gln Thr Glu Lys Leu Asp Thr Asn Ile Thr Glu Val
180 185 190
Asp Ala Ala Ser Val Tyr Thr Leu Pro Ala Gly Ala Asp Phe Leu Met
195 200 205
Cys Tyr Ser Val Ala Glu Gly Tyr Tyr Ser His Arg Glu Thr Val Asn
210 215 220
Gly Ser Trp Tyr Ile Gln Asp Leu Cys Glu Met Leu Gly Lys Tyr Gly
225 230 235 240
Ser Ser Leu Glu Phe Thr Glu Leu Leu Thr Leu Val Asn Arg Lys Val
245 250 255
Ser Gln Arg Arg Val Asp Phe Cys Lys Asp Pro Ser Ala Ile Gly Lys
260 265 270
Lys Gln Val Pro Cys Phe Ala Ser Met Leu Thr Lys Lys Leu His Phe
275 280 285
Phe Pro Lys Ser Asn
290
<210>24
<211>303
<212>PRT
<213> human (Homo sapiens)
<400>24
Met Ala Asp Asp Gln Gly Cys Ile Glu Glu Gln Gly Val Glu Asp Ser
1 5 10 15
Ala Asn Glu Asp Ser Val Asp Ala Lys Pro Asp Arg Ser Ser Phe Val
20 25 30
Pro Ser Leu Phe Ser Lys Lys Lys Lys Asn Val Thr Met Arg Ser Ile
35 40 45
Lys Thr Thr Arg Asp Arg Val Pro Thr Tyr Gln Tyr Asn Met Asn Phe
50 55 60
Glu Lys Leu Gly Lys Cys Ile Ile Ile Asn Asn Lys Asn Phe Asp Lys
65 70 75 80
Val Thr Gly Met Gly Val Arg Asn Gly Thr Asp Lys Asp Ala Glu Ala
85 90 95
Leu Phe Lys Cys Phe Arg Ser Leu Gly Phe Asp Val Ile Val Tyr Asn
100 105 110
Asp Cys Ser Cys Ala Lys Met Gln Asp Leu Leu Lys Lys Ala Ser Glu
115 120 125
Glu Asp His Thr Asn Ala Ala Cys Phe Ala Cys Ile Leu Leu Ser His
130 135 140
Gly Glu Glu Asn Val Ile Tyr Gly Lys Asp Gly Val Thr Pro Ile Lys
145 150155 160
Asp Leu Thr Ala His Phe Arg Gly Asp Arg Cys Lys Thr Leu Leu Glu
165 170 175
Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Arg Gly Thr Glu Leu Asp
180 185 190
Asp Gly Ile Gln Ala Asp Ser Gly Pro Ile Asn Asp Thr Asp Ala Asn
195 200 205
Pro Arg Tyr Lys Ile Pro Val Glu Ala Asp Phe Leu Phe Ala Tyr Ser
210 215 220
Thr Val Pro Gly Tyr Tyr Ser Trp Arg Ser Pro Gly Arg Gly Ser Trp
225 230 235 240
Phe Val Gln Ala Leu Cys Ser Ile Leu Glu Glu His Gly Lys Asp Leu
245 250 255
Glu Ile Met Gln Ile Leu Thr Arg Val Asn Asp Arg Val Ala Arg His
260 265 270
Phe Glu Ser Gln Ser Asp Asp Pro His Phe His Glu Lys Lys Gln Ile
275 280 285
Pro Cys Val Val Ser Met Leu Thr Lys Glu Leu Tyr Phe Ser Gln
290 295 300
<210>25
<211>479
<212>PRT
<213> human (Homo sapiens)
<400>25
Met Asp Phe Ser Arg Asn Leu Tyr Asp Ile Gly Glu Gln Leu Asp Ser
1 5 10 15
Glu Asp Leu Ala Ser Leu Lys Phe Leu Ser Leu Asp Tyr Ile Pro Gln
20 25 30
Arg Lys Gln Glu Pro Ile Lys Asp Ala Leu Met Leu Phe Gln Arg Leu
35 40 45
Gln Glu Lys Arg Met Leu Glu Glu Ser Asn Leu Ser Phe Leu Lys Glu
50 55 60
Leu Leu Phe Arg Ile Asn Arg Leu Asp Leu Leu Ile Thr Tyr Leu Asn
65 70 75 80
Thr Arg Lys Glu Glu Met Glu Arg Glu Leu Gln Thr Pro Gly Arg Ala
85 90 95
Gln Ile Ser Ala Tyr Arg Val Met Leu Tyr Gln Ile Ser Glu Glu Val
100 105 110
Ser Arg Ser Glu Leu Arg Ser Phe Lys Phe Leu Leu Gln Glu Glu Ile
115 120 125
Ser Lys Cys Lys Leu Asp Asp Asp Met Asn Leu Leu Asp Ile Phe Ile
130 135 140
Glu Met Glu Lys Arg Val Ile Leu Gly Glu Gly Lys Leu Asp Ile Leu
145 150 155 160
Lys Arg Val Cys Ala Gln Ile Asn Lys Ser Leu Leu Lys Ile Ile Asn
165 170 175
Asp Tyr Glu Glu Phe Ser Lys Glu Arg Ser Ser Ser Leu Glu Gly Ser
180 185 190
Pro Asp Glu Phe Ser Asn Gly Glu Glu Leu Cys Gly Val Met Thr Ile
195 200 205
Ser Asp Ser Pro Arg Glu Gln Asp Ser Glu Ser Gln Thr Leu Asp Lys
210 215 220
Val Tyr Gln Met Lys Ser Lys Pro Arg Gly Tyr Cys Leu Ile Ile Asn
225 230 235 240
Asn His Asn Phe Ala Lys Ala Arg Glu Lys Val Pro Lys Leu His Ser
245 250 255
Ile Arg Asp Arg Asn Gly Thr His Leu Asp Ala Gly Ala Leu Thr Thr
260 265 270
Thr Phe Glu Glu Leu His Phe Glu Ile Lys Pro His Asp Asp Cys Thr
275 280 285
Val Glu Gln Ile Tyr Glu Ile Leu Lys Ile Tyr Gln Leu Met Asp His
290 295 300
Ser Asn Met Asp Cys Phe Ile Cys Cys Ile Leu Ser His Gly Asp Lys
305 310 315 320
Gly Ile Ile Tyr Gly Thr Asp Gly Gln Glu Ala Pro Ile Tyr Glu Leu
325 330 335
Thr Ser Gln Phe Thr Gly Leu Lys Cys Pro Ser Leu Ala Gly Lys Pro
340 345 350
Lys Val Phe Phe Ile Gln Ala Cys Gln Gly Asp Asn Tyr Gln Lys Gly
355 360 365
Ile Pro Val Glu Thr Asp Ser Glu Glu Gln Pro Tyr Leu Glu Met Asp
370 375 380
Leu Ser Ser Pro Gln Thr Arg TyrIle Pro Asp Glu Ala Asp Phe Leu
385 390 395 400
Leu Gly Met Ala Thr Val Asn Asn Cys Val Ser Tyr Arg Asn Pro Ala
405 410 415
Glu Gly Thr Trp Tyr Ile Gln Ser Leu Cys Gln Ser Leu Arg Glu Arg
420 425 430
Cys Pro Arg Gly Asp Asp Ile Leu Thr Ile Leu Thr Glu Val Asn Tyr
435 440 445
Glu Val Ser Asn Lys Asp Asp Lys Lys Asn Met Gly Lys Gln Met Pro
450 455 460
Gln Pro Thr Phe Thr Leu Arg Lys Lys Leu Val Phe Pro Ser Asp
465 470 475
<210>26
<211>416
<212>PRT
<213> human (Homo sapiens)
<400>26
Met Asp Glu Ala Asp Arg Arg Leu Leu Arg Arg Cys Arg Leu Arg Leu
1 5 10 15
Val Glu Glu Leu Gln Val Asp Gln Leu Trp Asp Ala Leu Leu Ser Arg
20 25 30
Glu Leu Phe Arg Pro His Met Ile Glu Asp Ile Gln Arg Ala Gly Ser
35 40 45
Gly Ser Arg Arg Asp Gln Ala Arg Gln Leu Ile Ile Asp Leu Glu Thr
50 55 60
Arg Gly Ser Gln Ala Leu Pro Leu Phe Ile Ser Cys Leu Glu Asp Thr
65 70 75 80
Gly Gln Asp Met Leu Ala Ser Phe Leu Arg Thr Asn Arg Gln Ala Ala
85 90 95
Lys Leu Ser Lys Pro Thr Leu Glu Asn Leu Thr Pro Val Val Leu Arg
100 105 110
Pro Glu Ile Arg Lys Pro Glu Val Leu Arg Pro Glu Thr Pro Arg Pro
115 120 125
Val Asp Ile Gly Ser Gly Gly Phe Gly Asp Val Gly Ala Leu Glu Ser
130 135 140
Leu Arg Gly Asn Ala Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys
145 150 155 160
Gly His Cys LeuIle Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly
165 170 175
Leu Arg Thr Arg Thr Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg
180 185 190
Arg Phe Ser Ser Leu His Phe Met Val Glu Val Lys Gly Asp Leu Thr
195 200 205
Ala Lys Lys Met Val Leu Ala Leu Leu Glu Leu Ala Gln Gln Asp His
210 215 220
Gly Ala Leu Asp Cys Cys Val Val Val Ile Leu Ser His Gly Cys Gln
225 230 235 240
Ala Ser His Leu Gln Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys
245 250 255
Pro Val Ser Val Glu Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys
260 265 270
Pro Ser Leu Gly Gly Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly
275 280 285
Gly Glu Gln Lys Asp His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu
290 295 300
Asp Glu Ser Pro Gly Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln
305 310 315 320
Glu Gly Leu Arg Thr Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro
325 330 335
Thr Pro Ser Asp Ile Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val
340 345 350
Ser Trp Arg Asp Pro Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp
355 360 365
Asp Ile Phe Glu Gln Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu
370 375 380
Leu Arg Val Ala Asn Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met
385 390 395 400
Pro Gly Cys Phe Asn Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser
405 410 415
<210>27
<211>521
<212>PRT
<213> human (Homo sapiens)
<400>27
Met Lys Ser Gln Gly Gln His Trp Tyr Ser Ser Ser Asp Lys Asn Cys
1 5 10 15
Lys Val Ser Phe Arg Glu Lys Leu Leu Ile Ile Asp Ser Asn Leu Gly
20 25 30
Val Gln Asp Val Glu Asn Leu Lys Phe Leu Cys Ile Gly Leu Val Pro
35 40 45
Asn Lys Lys Leu Glu Lys Ser Ser Ser Ala Ser Asp Val Phe Glu His
50 55 60
Leu Leu Ala Glu Asp Leu Leu Ser Glu Glu Asp Pro Phe Phe Leu Ala
65 70 75 80
Glu Leu Leu Tyr Ile Ile Arg Gln Lys Lys Leu Leu Gln His Leu Asn
85 90 95
Cys Thr Lys Glu Glu Val Glu Arg Leu Leu Pro Thr Arg Gln Arg Val
100 105 110
Ser Leu Phe Arg Asn Leu Leu Tyr Glu Leu Ser Glu Gly Ile Asp Ser
115 120 125
Glu Asn Leu Lys Asp Met Ile Phe Leu Leu Lys Asp Ser Leu Pro Lys
130 135140
Thr Glu Met Thr Ser Leu Ser Phe Leu Ala Phe Leu Glu Lys Gln Gly
145 150 155 160
Lys Ile Asp Glu Asp Asn Leu Thr Cys Leu Glu Asp Leu Cys Lys Thr
165 170 175
Val Val Pro Lys Leu Leu Arg Asn Ile Glu Lys Tyr Lys Arg Glu Lys
180 185 190
Ala Ile Gln Ile Val Thr Pro Pro Val Asp Lys Glu Ala Glu Ser Tyr
195 200 205
Gln Gly Glu Glu Glu Leu Val Ser Gln Thr Asp Val Lys Thr Phe Leu
210 215 220
Glu Ala Leu Pro Gln Glu Ser Trp Gln Asn Lys His Ala Gly Ser Asn
225 230 235 240
Gly Asn Arg Ala Thr Asn Gly Ala Pro Ser Leu Val Ser Arg Gly Met
245 250 255
Gln Gly Ala Ser Ala Asn Thr Leu Asn Ser Glu Thr Ser Thr Lys Arg
260 265 270
Ala Ala Val Tyr Arg Met Asn Arg Asn His Arg Gly Leu Cys Val Ile
275 280 285
Val Asn Asn His Ser Phe Thr Ser Leu Lys Asp Arg Gln Gly Thr His
290 295 300
Lys Asp Ala Glu Ile Leu Ser His Val Phe Gln Trp Leu Gly Phe Thr
305 310 315 320
Val His Ile His Asn Asn Val Thr Lys Val Glu Met Glu Met Val Leu
325 330 335
Gln Lys Gln Lys Cys Asn Pro Ala His Ala Asp Gly Asp Cys Phe Val
340 345 350
Phe Cys Ile Leu Thr His Gly Arg Phe Gly Ala Val Tyr Ser Ser Asp
355 360 365
Glu Ala Leu Ile Pro Ile Arg Glu Ile Met Ser His Phe Thr Ala Leu
370 375 380
Gln Cys Pro Arg Leu Ala Glu Lys Pro Lys Leu Phe Phe Ile Gln Ala
385 390 395 400
Cys Gln Gly Glu Glu Ile Gln Pro Ser Val Ser Ile Glu Ala Asp Ala
405 410 415
Leu Asn Pro Glu Gln Ala Pro Thr Ser Leu GlnAsp Ser Ile Pro Ala
420 425 430
Glu Ala Asp Phe Leu Leu Gly Leu Ala Thr Val Pro Gly Tyr Val Ser
435 440 445
Phe Arg His Val Glu Glu Gly Ser Trp Tyr Ile Gln Ser Leu Cys Asn
450 455 460
His Leu Lys Lys Leu Val Pro Arg Met Leu Lys Phe Leu Glu Lys Thr
465 470 475 480
Met Glu Ile Arg Gly Arg Lys Arg Thr Val Trp Gly Ala Lys Gln Ile
485 490 495
Ser Ala Thr Ser Leu Pro Thr Ala Ile Ser Ala Gln Thr Pro Arg Pro
500 505 510
Pro Met Arg Arg Trp Ser Ser Val Ser
515 520
<210>28
<211>373
<212>PRT
<213> human (Homo sapiens)
<400>28
Met Ala Glu Asn Lys His Pro Asp Lys Pro Leu Lys Val Leu Glu Gln
15 10 15
Leu Gly Lys Glu Val Leu Thr Glu Tyr Leu Glu Lys Leu Val Gln Ser
20 25 30
Asn Val Leu Lys Leu Lys Glu Glu Asp Lys Gln Lys Phe Asn Asn Ala
35 40 45
Glu Arg Ser Asp Lys Arg Trp Val Phe Val Asp Ala Met Lys Lys Lys
50 55 60
His Ser Lys Val Gly Glu Met Leu Leu Gln Thr Phe Phe Ser Val Asp
65 70 75 80
Pro Gly Ser His His Gly Glu Ala Asn Leu Glu Met Glu Glu Pro Glu
85 90 95
Glu Ser Leu Asn Thr Leu Lys Leu Cys Ser Pro Glu Glu Phe Thr Arg
100 105 110
Leu Cys Arg Glu Lys Thr Gln Glu Ile Tyr Pro Ile Lys Glu Ala Asn
115 120 125
Gly Arg Thr Arg Lys Ala Leu Ile Ile Cys Asn Thr Glu Phe Lys His
130 135 140
Leu Ser Leu Arg Tyr Gly Ala Asn Phe Asp Ile Ile Gly Met Lys Gly
145 150 155 160
Leu Leu Glu Asp Leu Gly Tyr Asp Val Val Val Lys Glu Glu Leu Thr
165 170 175
Ala Glu Gly Met Glu Ser Glu Met Lys Asp Phe Ala Ala Leu Ser Glu
180 185 190
His Gln Thr Ser Asp Ser Thr Phe Leu Val Leu Met Ser His Gly Thr
195 200 205
Leu His Gly Ile Cys Gly Thr Met His Ser Glu Lys Thr Pro Asp Val
210 215 220
Leu Gln Tyr Asp Thr Ile Tyr Gln Ile Phe Asn Asn Cys His Cys Pro
225 230 235 240
Gly Leu Arg Asp Lys Pro Lys Val Ile Ile Val Gln Ala Cys Arg Gly
245 250 255
Gly Asn Ser Gly Glu Met Trp Ile Arg Glu Ser Ser Lys Pro Gln Leu
260 265 270
Cys Arg Gly Val Asp Leu Pro Arg Asn Met Glu Ala Asp Ala Val Lys
275 280 285
Leu Ser His ValGlu Lys Asp Phe Ile Ala Phe Tyr Ser Thr Thr Pro
290 295 300
His His Leu Ser Tyr Arg Asp Lys Thr Gly Gly Ser Tyr Phe Ile Thr
305 310 315 320
Arg Leu Ile Ser Cys Phe Arg Lys His Ala Cys Ser Cys His Leu Phe
325 330 335
Asp Ile Phe Leu Lys Val Gln Gln Ser Phe Glu Lys Ala Ser Ile His
340 345 350
Ser Gln Met Pro Thr Ile Asp Arg Ala Thr Leu Thr Arg Tyr Phe Tyr
355 360 365
Leu Phe Pro Gly Asn
370
<210>29
<211>341
<212>PRT
<213> human (Homo sapiens)
<400>29
Met Ala Asp Glu Lys Pro Ser Asn Gly Val Leu Val His Met Val Lys
1 5 10 15
Leu Leu Ile Lys Thr Phe Leu Asp Gly Ile Phe Asp Asp Leu Met Glu
20 25 30
Asn Asn Val Leu Asn Thr Asp Glu Ile His Leu Ile Gly Lys Cys Leu
35 40 45
Lys Phe Val Val Ser Asn Ala Glu Asn Leu Val Asp Asp Ile Thr Glu
50 55 60
Thr Ala Gln Thr Ala Gly Lys Ile Phe Arg Glu His Leu Trp Asn Ser
65 70 75 80
Lys Lys Gln Leu Ser Ser Asp Ile Ser Ser Asp Gly Glu Arg Glu Ala
85 90 95
Asn Met Pro Gly Leu Asn Ile Arg Asn Lys Glu Phe Asn Tyr Leu His
100 105 110
Asn Arg Asn Gly Ser Glu Leu Asp Leu Leu Gly Met Arg Asp Leu Leu
115 120 125
Glu Asn Leu Gly Tyr Ser Val Val Ile Lys Glu Asn Leu Thr Ala Gln
130 135 140
Glu Met Glu Thr Ala Leu Arg Gln Phe Ala Ala His Pro Glu His Gln
145 150 155 160
Ser Ser Asp Ser Thr Phe Leu Val Phe Met Ser His Ser Ile Leu Asn
165 170 175
Gly Ile Cys Gly Thr Lys His Trp Asp Gln Glu Pro Asp Val Leu His
180 185 190
Asp Asp Thr Ile Phe Glu Ile Phe Asn Asn Arg Asn Cys Gln Ser Leu
195 200 205
Lys Asp Lys Pro Lys Val Ile Ile Met Gln Ala Cys Arg Gly Asn Gly
210 215 220
Ala Gly Ile Val Trp Phe Thr Thr Asp Ser Gly Lys Ala Gly Ala Asp
225 230 235 240
Thr His Gly Arg Leu Leu Gln Gly Asn Ile Cys Asn Asp Ala Val Thr
245 250 255
Lys Ala His Val Glu Lys Asp Phe Ile Ala Phe Lys Ser Ser Thr Pro
260 265 270
His Asn Val Ser Trp Arg His Glu Thr Asn Gly Ser Val Phe Ile Ser
275 280 285
Gln Ile Ile Tyr Tyr Phe Arg Glu Tyr Ser Trp Ser His His Leu Glu
290 295 300
Glu Ile Phe Gln Lys Val Gln His Ser Phe Glu Thr Pro Asn Ile Leu
305 310 315 320
Thr Gln Leu Pro Thr Ile Glu Arg Leu Ser Met Thr Arg Tyr Phe Tyr
325 330 335
Leu Phe Pro Gly Asn
340
<210>30
<211>377
<212>PRT
<213> human (Homo sapiens)
<400>30
Met Ala Glu Asp Lys His Asn Lys Asn Pro Leu Lys Met Leu Glu Ser
1 5 10 15
Leu Gly Lys Glu Leu Ile Ser Gly Leu Leu Asp Asp Phe Val Glu Lys
20 25 30
Asn Val Leu Lys Leu Glu Glu Glu Glu Lys Lys Lys Ile Tyr Asp Ala
35 40 45
Lys Leu Gln Asp Lys Ala Arg Val Leu Val Asp Ser Ile Arg Gln Lys
50 55 60
Asn Gln Glu Ala Gly Gln Val Phe Val Gln Thr Phe Leu Asn Ile Asp
65 7075 80
Lys Asn Ser Thr Ser Ile Lys Ala Pro Glu Glu Thr Val Ala Gly Pro
85 90 95
Asp Glu Ser Val Gly Ser Ala Ala Thr Leu Lys Leu Cys Pro His Glu
100 105 110
Glu Phe Leu Lys Leu Cys Lys Glu Arg Ala Gly Glu Ile Tyr Pro Ile
115 120 125
Lys Glu Arg Lys Asp Arg Thr Arg Leu Ala Leu Ile Ile Cys Asn Thr
130 135 140
Glu Phe Asp His Met Pro Pro Arg Asn Gly Ala Ala Leu Asp Ile Leu
145 150 155 160
Gly Met Lys Gln Leu Leu Glu Gly Leu Gly Tyr Thr Val Glu Val Glu
165 170 175
Glu Lys Leu Thr Ala Arg Asp Met Glu Ser Val Leu Trp Lys Phe Ala
180 185 190
Ala Arg Glu Glu His Lys Ser Ser Asp Ser Thr Phe Leu Val Phe Met
195 200 205
Ser His Gly Ile Leu Asp Gly Ile Cys Gly Thr Met His Ser Glu Glu
210 215 220
Glu Pro Asp Val Leu Pro Tyr Asp Thr Ile Phe Arg Thr Phe Asn Asn
225 230 235 240
Arg Asn Cys Leu Ser Leu Lys Asp Lys Pro Lys Val Ile Ile Val Gln
245 250 255
Ala Cys Arg Gly Ala Asn Arg Gly Glu Leu Trp Val Ser Asp Ser Pro
260 265 270
Pro Ala Leu Ala Asp Ser Phe Ser Gln Ser Ser Glu Asn Leu Glu Glu
275 280 285
Asp Ala Val Tyr Lys Thr His Val Glu Lys Asp Phe Ile Ala Phe Cys
290 295 300
Ser Ser Thr Pro His Asn Val Ser Trp Arg Asp Ile Lys Lys Gly Ser
305 310 315 320
Leu Phe Ile Thr Arg Leu Ile Thr Cys Phe Gln Lys Tyr Ala Trp Cys
325 330 335
Cys His Leu Glu Glu Val Phe Arg Lys Val Gln Gln Ser Phe Glu Lys
340 345 350
Pro Asn Val Lys Ala Gln Met Pro Thr Val Glu Arg Leu Ser Met Thr
355 360 365
Arg Tyr Phe Tyr Leu Phe Pro Gly Asn
370 375
<210>31
<211>242
<212>PRT
<213> human (Homo sapiens)
<400>31
Met Ser Asn Pro Arg Ser Leu Glu Glu Glu Lys Tyr Asp Met Ser Gly
1 5 10 15
Ala Arg Leu Ala Leu Ile Leu Cys Val Thr Lys Ala Arg Glu Gly Ser
20 25 30
Glu Glu Asp Leu Asp Ala Leu Glu His Met Phe Arg Gln Leu Arg Phe
35 40 45
Glu Ser Thr Met Lys Arg Asp Pro Thr Ala Glu Gln Phe Gln Glu Glu
50 55 60
Leu Glu Lys Phe Gln Gln Ala Ile Asp Ser Arg Glu Asp Pro Val Ser
65 70 75 80
Cys Ala Phe Val Val Leu Met Ala His Gly Arg Glu Gly Phe Leu Lys
85 90 95
Gly Glu Asp Gly Glu Met Val Lys Leu Glu Asn Leu Phe Glu Ala Leu
100 105 110
Asn Asn Lys Asn Cys Gln Ala Leu Arg Ala Lys Pro Lys Val Tyr Ile
115 120 125
Ile Gln Ala Cys Arg Gly Glu Gln Arg Asp Pro Gly Glu Thr Val Gly
130 135 140
Gly Asp Glu Ile Val Met Val Ile Lys Asp Ser Pro Gln Thr Ile Pro
145 150 155 160
Thr Tyr Thr Asp Ala Leu His Val Tyr Ser Thr Val Glu Gly Tyr Ile
165 170 175
Ala Tyr Arg His Asp Gln Lys Gly Ser Cys Phe Ile Gln Thr Leu Val
180 185 190
Asp Val Phe Thr Lys Arg Lys Gly His Ile Leu Glu Leu Leu Thr Glu
195 200 205
Val Thr Arg Arg Met Ala Glu Ala Glu Leu Val Gln Glu Gly Lys Ala
210 215 220
Arg Lys Thr Asn Pro Glu Ile Gln Ser Thr Leu Arg Lys Arg Leu Tyr
225 230 235 240
Leu Gln
<210>32
<211>480
<212>PRT
<213> human (Homo sapiens)
<400>32
Met Ile Arg Ala Ala Pro Pro Pro Leu Phe Leu Leu Leu Leu Leu Leu
1 5 10 15
Leu Leu Leu Val Ser Trp Ala Ser Arg Gly Glu Ala Ala Pro Asp Gln
20 25 30
Asp Glu Ile Gln Arg Leu Pro Gly Leu Ala Lys Gln Pro Ser Phe Arg
35 40 45
Gln Tyr Ser Gly Tyr Leu Lys Gly Ser Gly Ser Lys His Leu His Tyr
50 55 60
Trp Phe Val Glu Ser Gln Lys Asp Pro Glu Asn Ser Pro Val Val Leu
65 70 75 80
Trp Leu Asn Gly Gly Pro Gly Cys Ser Ser Leu Asp Gly Leu Leu Thr
85 90 95
Glu His Gly Pro Phe LeuVal Gln Pro Asp Gly Val Thr Leu Glu Tyr
100 105 110
Asn Pro Tyr Ser Trp Asn Leu Ile Ala Asn Val Leu Tyr Leu Glu Ser
115 120 125
Pro Ala Gly Val Gly Phe Ser Tyr Ser Asp Asp Lys Phe Tyr Ala Thr
130 135 140
Asn Asp Thr Glu Val Ala Gln Ser Asn Phe Glu Ala Leu Gln Asp Phe
145 150 155 160
Phe Arg Leu Phe Pro Glu Tyr Lys Asn Asn Lys Leu Phe Leu Thr Gly
165 170 175
Glu Ser Tyr Ala Gly Ile Tyr Ile Pro Thr Leu Ala Val Leu Val Met
180 185 190
Gln Asp Pro Ser Met Asn Leu Gln Gly Leu Ala Val Gly Asn Gly Leu
195 200 205
Ser Ser Tyr Glu Gln Asn Asp Asn Ser Leu Val Tyr Phe Ala Tyr Tyr
210 215 220
His Gly Leu Leu Gly Asn Arg Leu Trp Ser Ser Leu Gln Thr His Cys
225 230 235 240
Cys Ser Gln Asn Lys Cys Asn Phe Tyr Asp Asn Lys Asp Leu Glu Cys
245 250 255
Val Thr Asn Leu Gln Glu Val Ala Arg Ile Val Gly Asn Ser Gly Leu
260 265 270
Asn Ile Tyr Asn Leu Tyr Ala Pro Cys Ala Gly Gly Val Pro Ser His
275 280 285
Phe Arg Tyr Glu Lys Asp Thr Val Val Val Gln Asp Leu Gly Asn Ile
290 295 300
Phe Thr Arg Leu Pro Leu Lys Arg Met Trp His Gln Ala Leu Leu Arg
305 310 315 320
Ser Gly Asp Lys Val Arg Met Asp Pro Pro Cys Thr Asn Thr Thr Ala
325 330 335
Ala Ser Thr Tyr Leu Asn Asn Pro Tyr Val Arg Lys Ala Leu Asn Ile
340 345 350
Pro Glu Gln Leu Pro Gln Trp Asp Met Cys Asn Phe Leu Val Asn Leu
355 360 365
Gln Tyr Arg Arg Leu Tyr Arg Ser Met Asn Ser Gln Tyr Leu Lys Leu
370375 380
Leu Ser Ser Gln Lys Tyr Gln Ile Leu Leu Tyr Asn Gly Asp Val Asp
385 390 395 400
Met Ala Cys Asn Phe Met Gly Asp Glu Trp Phe Val Asp Ser Leu Asn
405 410 415
Gln Lys Met Glu Val Gln Arg Arg Pro Trp Leu Val Lys Tyr Gly Asp
420 425 430
Ser Gly Glu Gln Ile Ala Gly Phe Val Lys Glu Phe Ser His Ile Ala
435 440 445
Phe Leu Thr Ile Lys Gly Ala Gly His Met Val Pro Thr Asp Lys Pro
450 455 460
Leu Ala Ala Phe Thr Met Phe Ser Arg Phe Leu Asn Lys Gln Pro Tyr
465 470 475 480
<210>33
<211>339
<212>PRT
<213> human (Homo sapiens)
<400>33
Met Trp Gln Leu Trp Ala Ser Leu Cys Cys Leu Leu Val Leu Ala Asn
1 5 1015
Ala Arg Ser Arg Pro Ser Phe His Pro Leu Ser Asp Glu Leu Val Asn
20 25 30
Tyr Val Asn Lys Arg Asn Thr Thr Trp Gln Ala Gly His Asn Phe Tyr
35 40 45
Asn Val Asp Met Ser Tyr Leu Lys Arg Leu Cys Gly Thr Phe Leu Gly
50 55 60
Gly Pro Lys Pro Pro Gln Arg Val Met Phe Thr Glu Asp Leu Lys Leu
65 70 75 80
Pro Ala Ser Phe Asp Ala Arg Glu Gln Trp Pro Gln Cys Pro Thr Ile
85 90 95
Lys Glu Ile Arg Asp Gln Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly
100 105 110
Ala Val Glu Ala Ile Ser Asp Arg Ile Cys Ile His Thr Asn Ala His
115 120 125
Val Ser Val Glu Val Ser Ala Glu Asp Leu Leu Thr Cys Cys Gly Ser
130 135 140
Met Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pro Ala Glu Ala Trp Asn
145150 155 160
Phe Trp Thr Arg Lys Gly Leu Val Ser Gly Gly Leu Tyr Glu Ser His
165 170 175
Val Gly Cys Arg Pro Tyr Ser Ile Pro Pro Cys Glu His His Val Asn
180 185 190
Gly Ser Arg Pro Pro Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Ser
195 200 205
Lys Ile Cys Glu Pro Gly Tyr Ser Pro Thr Tyr Lys Gln Asp Lys His
210 215 220
Tyr Gly Tyr Asn Ser Tyr Ser Val Ser Asn Ser Glu Lys Asp Ile Met
225 230 235 240
Ala Glu Ile Tyr Lys Asn Gly Pro Val Glu Gly Ala Phe Ser Val Tyr
245 250 255
Ser Asp Phe Leu Leu Tyr Lys Ser Gly Val Tyr Gln His Val Thr Gly
260 265 270
Glu Met Met Gly Gly His Ala Ile Arg Ile Leu Gly Trp Gly Val Glu
275 280 285
Asn Gly Thr Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Thr AspTrp
290 295 300
Gly Asp Asn Gly Phe Phe Lys Ile Leu Arg Gly Gln Asp His Cys Gly
305 310 315 320
Ile Glu Ser Glu Val Val Ala Gly Ile Pro Arg Thr Asp Gln Tyr Trp
325 330 335
Glu Lys Ile
<210>34
<211>463
<212>PRT
<213> human (Homo sapiens)
<400>34
Met Gly Ala Gly Pro Ser Leu Leu Leu Ala Ala Leu Leu Leu Leu Leu
1 5 10 15
Ser Gly Asp Gly Ala Val Arg Cys Asp Thr Pro Ala Asn Cys Thr Tyr
20 25 30
Leu Asp Leu Leu Gly Thr Trp Val Phe Gln Val Gly Ser Ser Gly Ser
35 40 45
Gln Arg Asp Val Asn Cys Ser Val Met Gly Pro Gln Glu Lys Lys Val
50 55 60
Val Val Tyr Leu Gln Lys LeuAsp Thr Ala Tyr Asp Asp Leu Gly Asn
65 70 75 80
Ser Gly His Phe Thr Ile Ile Tyr Asn Gln Gly Phe Glu Ile Val Leu
85 90 95
Asn Asp Tyr Lys Trp Phe Ala Phe Phe Lys Tyr Lys Glu Glu Gly Ser
100 105 110
Lys Val Thr Thr Tyr Cys Asn Glu Thr Met Thr Gly Trp Val His Asp
115 120 125
Val Leu Gly Arg Asn Trp Ala Cys Phe Thr Gly Lys Lys Val Gly Thr
130 135 140
Ala Ser Glu Asn Val Tyr Val Asn Ile Ala His Leu Lys Asn Ser Gln
145 150 155 160
Glu Lys Tyr Ser Asn Arg Leu Tyr Lys Tyr Asp His Asn Phe Val Lys
165 170 175
Ala Ile Asn Ala Ile Gln Lys Ser Trp Thr Ala Thr Thr Tyr Met Glu
180 185 190
Tyr Glu Thr Leu Thr Leu Gly Asp Met Ile Arg Arg Ser Gly Gly His
195 200 205
Ser Arg Lys Ile Pro Arg Pro Lys Pro Ala Pro Leu Thr Ala Glu Ile
210 215 220
Gln Gln Lys Ile Leu His Leu Pro Thr Ser Trp Asp Trp Arg Asn Val
225 230 235 240
His Gly Ile Asn Phe Val Ser Pro Val Arg Asn Gln Ala Ser Cys Gly
245 250 255
Ser Cys Tyr Ser Phe Ala Ser Met Gly Met Leu Glu Ala Arg Ile Arg
260 265 270
Ile Leu Thr Asn Asn Ser Gln Thr Pro Ile Leu Ser Pro Gln Glu Val
275 280 285
Val Ser Cys Ser Gln Tyr Ala Gln Gly Cys Glu Gly Gly Phe Pro Tyr
290 295 300
Leu Ile Ala Gly Lys Tyr Ala Gln Asp Phe Gly Leu Val Glu Glu Ala
305 310 315 320
Cys Phe Pro Tyr Thr Gly Thr Asp Ser Pro Cys Lys Met Lys Glu Asp
325 330 335
Cys Phe Arg Tyr Tyr Ser Ser Glu Tyr His Tyr Val Gly Gly Phe Tyr
340345 350
Gly Gly Cys Asn Glu Ala Leu Met Lys Leu Glu Leu Val His His Gly
355 360 365
Pro Met Ala Val Ala Phe Glu Val Tyr Asp Asp Phe Leu His Tyr Lys
370 375 380
Lys Gly Ile Tyr His His Thr Gly Leu Arg Asp Pro Phe Asn Pro Phe
385 390 395 400
Glu Leu Thr Asn His Ala Val Leu Leu Val Gly Tyr Gly Thr Asp Ser
405 410 415
Ala Ser Gly Met Asp Tyr Trp Ile Val Lys Asn Ser Trp Gly Thr Gly
420 425 430
Trp Gly Glu Asn Gly Tyr Phe Arg Ile Arg Arg Gly Thr Asp Glu Cys
435 440 445
Ala Ile Glu Ser Ile Ala Val Ala Ala Thr Pro Ile Pro Lys Leu
450 455 460
<210>35
<211>412
<212>PRT
<213> human (Homo sapiens)
<400>35
Met Gln Pro Ser Ser Leu Leu Pro Leu Ala Leu Cys Leu Leu Ala Ala
1 5 10 15
Pro Ala Ser Ala Leu Val Arg Ile Pro Leu His Lys Phe Thr Ser Ile
20 25 30
Arg Arg Thr Met Ser Glu Val Gly Gly Ser Val Glu Asp Leu Ile Ala
35 40 45
Lys Gly Pro Val Ser Lys Tyr Ser Gln Ala Val Pro Ala Val Thr Glu
50 55 60
Gly Pro Ile Pro Glu Val Leu Lys Asn Tyr Met Asp Ala Gln Tyr Tyr
65 70 75 80
Gly Glu Ile Gly Ile Gly Thr Pro Pro Gln Cys Phe Thr Val Val Phe
85 90 95
Asp Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Ile His Cys Lys Leu
100 105 110
Leu Asp Ile Ala Cys Trp Ile His His Lys Tyr Asn Ser Asp Lys Ser
115 120 125
Ser Thr Tyr Val Lys Asn Gly Thr Ser Phe Asp Ile His Tyr Gly Ser
130 135 140
Gly Ser Leu Ser Gly Tyr Leu Ser Gln Asp Thr Val Ser Val Pro Cys
145 150 155 160
Gln Ser Ala Ser Ser Ala Ser Ala Leu Gly Gly Val Lys Val Glu Arg
165 170 175
Gln Val Phe Gly Glu Ala Thr Lys Gln Pro Gly Ile Thr Phe Ile Ala
180 185 190
Ala Lys Phe Asp Gly Ile Leu Gly Met Ala Tyr Pro Arg Ile Ser Val
195 200 205
Asn Asn Val Leu Pro Val Phe Asp Asn Leu Met Gln Gln Lys Leu Val
210 215 220
Asp Gln Asn Ile Phe Ser Phe Tyr Leu Ser Arg Asp Pro Asp Ala Gln
225 230 235 240
Pro Gly Gly Glu Leu Met Leu Gly Gly Thr Asp Ser Lys Tyr Tyr Lys
245 250 255
Gly Ser Leu Ser Tyr Leu Asn Val Thr Arg Lys Ala Tyr Trp Gln Val
260 265 270
His Leu Asp Gln Val Glu Val Ala Ser Gly Leu Thr Leu Cys Lys Glu
275 280 285
Gly Cys Glu Ala Ile Val Asp Thr Gly Thr Ser Leu Met Val Gly Pro
290 295 300
Val Asp Glu Val Arg Glu Leu Gln Lys Ala Ile Gly Ala Val Pro Leu
305 310 315 320
Ile Gln Gly Glu Tyr Met Ile Pro Cys Glu Lys Val Ser Thr Leu Pro
325 330 335
Ala Ile Thr Leu Lys Leu Gly Gly Lys Gly Tyr Lys Leu Ser Pro Glu
340 345 350
Asp Tyr Thr Leu Lys Val Ser Gln Ala Gly Lys Thr Leu Cys Leu Ser
355 360 365
Gly Phe Met Gly Met Asp Ile Pro Pro Pro Ser Gly Pro Leu Trp Ile
370 375 380
Leu Gly Asp Val Phe Ile Gly Arg Tyr Tyr Thr Val Phe Asp Arg Asp
385 390 395 400
Asn Asn Arg Val Gly Phe Ala Glu Ala Ala Arg Leu
405 410
<210>36
<211>401
<212>PRT
<213> human (Homo sapiens)
<400>36
Met Lys Thr Leu Leu Leu Leu Leu Leu Val Leu Leu Glu Leu Gly Glu
1 5 10 15
Ala Gln Gly Ser Leu His Arg Val Pro Leu Arg Arg His Pro Ser Leu
20 25 30
Lys Lys Lys Leu Arg Ala Arg Ser Gln Leu Ser Glu Phe Trp Lys Ser
35 40 45
His Asn Leu Asp Met Ile Gln Phe Thr Glu Ser Cys Ser Met Asp Gln
50 55 60
Ser Ala Lys Glu Pro Leu Ile Asn Tyr Leu Asp Met Glu Tyr Phe Gly
65 70 75 80
Thr Ile Ser Ile Gly Ser Pro Pro Gln Asn Phe Thr Val Ile Phe Asp
85 90 95
Thr Gly Ser Ser Asn Leu Trp Val Pro Ser Val Tyr Cys Thr Ser Pro
100 105 110
Ala Cys Lys Thr His Ser Arg Phe Gln Pro Ser Gln Ser Ser Thr Tyr
115 120 125
Ser Gln Pro Gly Gln Ser Phe Ser Ile Gln Tyr Gly Thr Gly Ser Leu
130 135 140
Ser Gly Ile Ile Gly Ala Asp Gln Val Ser Ala Phe Ala Thr Gln Val
145 150 155 160
Glu Gly Leu Thr Val Val Gly Gln Gln Phe Gly Glu Ser Val Thr Glu
165 170 175
Pro Gly Gln Thr Phe Val Asp Ala Glu Phe Asp Gly Ile Leu Gly Leu
180 185 190
Gly Tyr Pro Ser Leu Ala Val Gly Gly Val Thr Pro Val Phe Asp Asn
195 200 205
Met Met Ala Gln Asn Leu Val Asp Leu Pro Met Phe Ser Val Tyr Met
210 215 220
Ser Ser Asn Pro Glu Gly Gly Ala Gly Ser Glu Leu Ile Phe Gly Gly
225 230 235 240
Tyr Asp His Ser His Phe Ser Gly Ser Leu Asn Trp Val Pro Val Thr
245 250 255
Lys GlnAla Tyr Trp Gln Ile Ala Leu Asp Asn Ile Gln Val Gly Gly
260 265 270
Thr Val Met Phe Cys Ser Glu Gly Cys Gln Ala Ile Val Asp Thr Gly
275 280 285
Thr Ser Leu Ile Thr Gly Pro Ser Asp Lys Ile Lys Gln Leu Gln Asn
290 295 300
Ala Ile Gly Ala Ala Pro Val Asp Gly Glu Tyr Ala Val Glu Cys Ala
305 310 315 320
Asn Leu Asn Val Met Pro Asp Val Thr Phe Thr Ile Asn Gly Val Pro
325 330 335
Tyr Thr Leu Ser Pro Thr Ala Tyr Thr Leu Leu Asp Phe Val Asp Gly
340 345 350
Met Gln Phe Cys Ser Ser Gly Phe Gln Gly Leu Asp Ile His Pro Pro
355 360 365
Ala Gly Pro Leu Trp Ile Leu Gly Asp Val Phe Ile Arg Gln Phe Tyr
370 375 380
Ser Val Phe Asp Arg Gly Asn Asn Arg Val Gly Leu Ala Pro Ala Val
385 390 395 400
Pro
<210>37
<211>484
<212>PRT
<213> human (Homo sapiens)
<400>37
Met Ala Pro Trp Leu Gln Leu Leu Ser Leu Leu Gly Leu Leu Pro Gly
1 5 10 15
Ala Val Ala Ala Pro Ala Gln Pro Arg Ala Ala Ser Phe Gln Ala Trp
20 25 30
Gly Pro Pro Ser Pro Glu Leu Leu Ala Pro Thr Arg Phe Ala Leu Glu
35 40 45
Met Phe Asn Arg Gly Arg Ala Ala Gly Thr Arg Ala Val Leu Gly Leu
50 55 60
Val Arg Gly Arg Val Arg Arg Ala Gly Gln Gly Ser Leu Tyr Ser Leu
65 70 75 80
Glu Ala Thr Leu Glu Glu Pro Pro Cys Asn Asp Pro Met Val Cys Arg
85 90 95
Leu Pro Val Ser Lys Lys Thr Leu Leu Cys Ser Phe Gln Val Leu Asp
100 105110
Glu Leu Gly Arg His Val Leu Leu Arg Lys Asp Cys Gly Pro Val Asp
115 120 125
Thr Lys Val Pro Gly Ala Gly Glu Pro Lys Ser Ala Phe Thr Gln Gly
130 135 140
Ser Ala Met Ile Ser Ser Leu Ser Gln Asn His Pro Asp Asn Arg Asn
145 150 155 160
Glu Thr Phe Ser Ser Val Ile Ser Leu Leu Asn Glu Asp Pro Leu Ser
165 170 175
Gln Asp Leu Pro Val Lys Met Ala Ser Ile Phe Lys Asn Phe Val Ile
180 185 190
Thr Tyr Asn Arg Thr Tyr Glu Ser Lys Glu Glu Ala Arg Trp Arg Leu
195 200 205
Ser Val Phe Val Asn Asn Met Val Arg Ala Gln Lys Ile Gln Ala Leu
210 215 220
Asp Arg Gly Thr Ala Gln Tyr Gly Val Thr Lys Phe Ser Asp Leu Thr
225 230 235 240
Glu Glu Glu Phe Arg Thr Ile Tyr Leu Asn Thr Leu Leu Arg Lys Glu
245 250 255
Pro Gly Asn Lys Met Lys Gln Ala Lys Ser Val Gly Asp Leu Ala Pro
260 265 270
Pro Glu Trp Asp Trp Arg Ser Lys Gly Ala Val Thr Lys Val Lys Asp
275 280 285
Gln Gly Met Cys Gly Ser Cys Trp Ala Phe Ser Val Thr Gly Asn Val
290 295 300
Glu Gly Gln Trp Phe Leu Asn Gln Gly Thr Leu Leu Ser Leu Ser Glu
305 310 315 320
Gln Glu Leu Leu Asp Cys Asp Lys Met Asp Lys Ala Cys Met Gly Gly
325 330 335
Leu Pro Ser Asn Ala Tyr Ser Ala Ile Lys Asn Leu Gly Gly Leu Glu
340 345 350
Thr Glu Asp Asp Tyr Ser Tyr Gln Gly His Met Gln Ser Cys Asn Phe
355 360 365
Ser Ala Glu Lys Ala Lys Val Tyr Ile Asn Asp Ser Val Glu Leu Ser
370 375 380
Gln Asn Glu Gln Lys Leu Ala Ala Trp Leu Ala Lys Arg Gly Pro Ile
385 390 395 400
Ser Val Ala Ile Asn Ala Phe Gly Met Gln Phe Tyr Arg His Gly Ile
405 410 415
Ser Arg Pro Leu Arg Pro Leu Cys Ser Pro Trp Leu Ile Asp His Ala
420 425 430
Val Leu Leu Val Gly Tyr Gly Asn Arg Ser Asp Val Pro Phe Trp Ala
435 440 445
Ile Lys Asn Ser Trp Gly Thr Asp Trp Gly Glu Lys Gly Tyr Tyr Tyr
450 455 460
Leu His Arg Gly Ser Gly Ala Cys Gly Val Asn Thr Met Ala Ser Ser
465 470 475 480
Ala Val Val Asp
<210>38
<211>255
<212>PRT
<213> human (Homo sapiens)
<400>38
Met Gln Pro Leu Leu Leu Leu Leu Ala Phe Leu Leu Pro Thr Gly Ala
1 5 10 15
Glu Ala Gly Glu Ile Ile Gly Gly Arg Glu Ser Arg Pro His Ser Arg
20 25 30
Pro Tyr Met Ala Tyr Leu Gln Ile Gln Ser Pro Ala Gly Gln Ser Arg
35 40 45
Cys Gly Gly Phe Leu Val Arg Glu Asp Phe Val Leu Thr Ala Ala His
50 55 60
Cys Trp Gly Ser Asn Ile Asn Val Thr Leu Gly Ala His Asn Ile Gln
65 70 75 80
Arg Arg Glu Asn Thr Gln Gln His Ile Thr Ala Arg Arg Ala Ile Arg
85 90 95
His Pro Gln Tyr Asn Gln Arg Thr Ile Gln Asn Asp Ile Met Leu Leu
100 105 110
Gln Leu Ser Arg Arg Val Arg Arg Asn Arg Asn Val Asn Pro Val Ala
115 120 125
Leu Pro Arg Ala Gln Glu Gly Leu Arg Pro Gly Thr Leu Cys Thr Val
130 135 140
Ala Gly Trp Gly Arg Val Ser Met Arg Arg Gly Thr Asp Thr Leu Arg
145 150 155 160
Glu Val Gln Leu Arg Val Gln Arg Asp Arg Gln Cys Leu Arg Ile Phe
165 170 175
Gly Ser Tyr Asp Pro Arg Arg Gln Ile Cys Val Gly Asp Arg Arg Glu
180 185 190
Arg Lys Ala Ala Phe Lys Gly Asp Ser Gly Gly Pro Leu Leu Cys Asn
195 200 205
Asn Val Ala His Gly Ile Val Ser Tyr Gly Lys Ser Ser Gly Val Pro
210 215 220
Pro Glu Val Phe Thr Arg Val Ser Ser Phe Leu Pro Trp Ile Arg Thr
225 230 235 240
Thr Met Arg Ser Phe Lys Leu Leu Asp Gln Met Glu Thr Pro Leu
245 250 255
<210>39
<211>335
<212>PRT
<213> human (Homo sapiens)
<400>39
Met Trp Ala Thr Leu Pro Leu Leu Cys Ala Gly Ala Trp Leu Leu Gly
1 5 10 15
Val Pro Val Cys Gly Ala Ala Glu Leu Cys Val Asn Ser Leu Glu Lys
20 25 30
Phe His Phe Lys Ser Trp Met Ser Lys His Arg Lys Thr Tyr Ser Thr
35 40 45
Glu Glu Tyr His His Arg Leu Gln Thr Phe Ala Ser Asn Trp Arg Lys
50 55 60
Ile Asn Ala His Asn Asn Gly Asn His Thr Phe Lys Met Ala Leu Asn
65 70 75 80
Gln Phe Ser Asp Met Ser Phe Ala Glu Ile Lys His Lys Tyr Leu Trp
85 90 95
Ser Glu Pro Gln Asn Cys Ser Ala Thr Lys Ser Asn Tyr Leu Arg Gly
100 105 110
Thr Gly Pro Tyr Pro Pro Ser Val Asp Trp Arg Lys Lys Gly Asn Phe
115 120 125
Val Ser Pro Val Lys Asn Gln Gly Ala Cys Gly Ser Cys Trp Thr Phe
130 135 140
Ser Thr Thr Gly Ala Leu Glu Ser Ala Ile Ala Ile Ala Thr Gly Lys
145 150155 160
Met Leu Ser Leu Ala Glu Gln Gln Leu Val Asp Cys Ala Gln Asp Phe
165 170 175
Asn Asn His Gly Cys Gln Gly Gly Leu Pro Ser Gln Ala Phe Glu Tyr
180 185 190
Ile Leu Tyr Asn Lys Gly Ile Met Gly Glu Asp Thr Tyr Pro Tyr Gln
195 200 205
Gly Lys Asp Gly Tyr Cys Lys Phe Gln Pro Gly Lys Ala Ile Gly Phe
210 215 220
Val Lys Asp Val Ala Asn Ile Thr Ile Tyr Asp Glu Glu Ala Met Val
225 230 235 240
Glu Ala Val Ala Leu Tyr Asn Pro Val Ser Phe Ala Phe Glu Val Thr
245 250 255
Gln Asp Phe Met Met Tyr Arg Thr Gly Ile Tyr Ser Ser Thr Ser Cys
260 265 270
His Lys Thr Pro Asp Lys Val Asn His Ala Val Leu Ala Val Gly Tyr
275 280 285
Gly Glu Lys Asn Gly Ile Pro Tyr Trp Ile Val Lys Asn Ser Trp Gly
290 295 300
Pro Gln Trp Gly Met Asn Gly Tyr Phe Leu Ile Glu Arg Gly Lys Asn
305 310 315 320
Met Cys Gly Leu Ala Ala Cys Ala Ser Tyr Pro Ile Pro Leu Val
325 330 335
<210>40
<211>329
<212>PRT
<213> human (Homo sapiens)
<400>40
Met Trp Gly Leu Lys Val Leu Leu Leu Pro Val Val Ser Phe Ala Leu
1 5 10 15
Tyr Pro Glu Glu Ile Leu Asp Thr His Trp Glu Leu Trp Lys Lys Thr
20 25 30
His Arg Lys Gln Tyr Asn Asn Lys Val Asp Glu Ile Ser Arg Arg Leu
35 40 45
Ile Trp Glu Lys Asn Leu Lys Tyr Ile Ser Ile His Asn Leu Glu Ala
50 55 60
Ser Leu Gly Val His Thr Tyr Glu Leu Ala Met Asn His Leu Gly Asp
6570 75 80
Met Thr Ser Glu Glu Val Val Gln Lys Met Thr Gly Leu Lys Val Pro
85 90 95
Leu Ser His Ser Arg Ser Asn Asp Thr Leu Tyr Ile Pro Glu Trp Glu
100 105 110
Gly Arg Ala Pro Asp Ser Val Asp Tyr Arg Lys Lys Gly Tyr Val Thr
115 120 125
Pro Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ser
130 135 140
Val Gly Ala Leu Glu Gly Gln Leu Lys Lys Lys Thr Gly Lys Leu Leu
145 150 155 160
Asn Leu Ser Pro Gln Asn Leu Val Asp Cys Val Ser Glu Asn Asp Gly
165 170 175
Cys Gly Gly Gly Tyr Met Thr Asn Ala Phe Gln Tyr Val Gln Lys Asn
180 185 190
Arg Gly Ile Asp Ser Glu Asp Ala Tyr Pro Tyr Val Gly Gln Glu Glu
195 200 205
Ser Cys Met Tyr Asn Pro Thr Gly Lys Ala Ala Lys Cys Arg Gly Tyr
210 215 220
Arg Glu Ile Pro Glu Gly Asn Glu Lys Ala Leu Lys Arg Ala Val Ala
225 230 235 240
Arg Val Gly Pro Val Ser Val Ala Ile Asp Ala Ser Leu Thr Ser Phe
245 250 255
Gln Phe Tyr Ser Lys Gly Val Tyr Tyr Asp Glu Ser Cys Asn Ser Asp
260 265 270
Asn Leu Asn His Ala Val Leu Ala Val Gly Tyr Gly Ile Gln Lys Gly
275 280 285
Asn Lys His Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asn
290 295 300
Lys Gly Tyr Ile Leu Met Ala Arg Asn Lys Asn Asn Ala Cys Gly Ile
305 310 315 320
Ala Asn Leu Ala Ser Phe Pro Lys Met
325
<210>41
<211>333
<212>PRT
<213> human (Homo sapiens)
<400>41
Met Asn Pro Thr Leu Ile Leu Ala Ala Phe Cys Leu Gly Ile Ala Ser
1 5 10 15
Ala Thr Leu Thr Phe Asp His Ser Leu Glu Ala Gln Trp Thr Lys Trp
20 25 30
Lys Ala Met His Asn Arg Leu Tyr Gly Met Asn Glu Glu Gly Trp Arg
35 40 45
Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gln
50 55 60
Glu Tyr Arg Glu Gly Lys His Ser Phe Thr Met Ala Met Asn Ala Phe
65 70 75 80
Gly Asp Met Thr Ser Glu Glu Phe Arg Gln Val Met Asn Gly Phe Gln
85 90 95
Asn Arg Lys Pro Arg Lys Gly Lys Val Phe Gln Glu Pro Leu Phe Tyr
100 105 110
Glu Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro
115 120 125
Val Lys Asn Gln Gly Gln Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr
130 135 140
Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Thr Gly Arg Leu Ile Ser
145 150 155 160
Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Gly Pro Gln Gly Asn Glu
165 170 175
Gly Cys Asn Gly Gly Leu Met Asp Tyr Ala Phe Gln Tyr Val Gln Asp
180 185 190
Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pro Tyr Glu Ala Thr Glu
195 200 205
Glu Ser Cys Lys Tyr Asn Pro Lys Tyr Ser Val Ala Asn Asp Thr Gly
210 215 220
Phe Val Asp Ile Pro Lys Gln Glu Lys Ala Leu Met Lys Ala Val Ala
225 230 235 240
Thr Val Gly Pro Ile Ser Val Ala Ile Asp Ala Gly His Glu Ser Phe
245 250 255
Leu Phe Tyr Lys Glu Gly Ile Tyr Phe Glu Pro Asp Cys Ser Ser Glu
260 265 270
Asp Met Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Ser Thr
275 280 285
Glu Ser Asp Asn Asn Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly Glu
290 295 300
Glu Trp Gly Met Gly Gly Tyr Val Lys Met Ala Lys Asp Arg Arg Asn
305 310 315 320
His Cys Gly Ile Ala Ser Ala Ala Ser Tyr Pro Thr Val
325 330
<210>42
<211>334
<212>PRT
<213> human (Homo sapiens)
<400>42
Met Asn Leu Ser Leu Val Leu Ala Ala Phe Cys Leu Gly Ile Ala Ser
1 5 10 15
Ala Val Pro Lys Phe Asp Gln Asn Leu Asp Thr Lys Trp Tyr Gln Trp
20 25 30
Lys Ala Thr His Arg Arg Leu Tyr Gly Ala Asn Glu Glu Gly Trp Arg
35 40 45
Arg Ala Val Trp Glu Lys Asn Met Lys Met Ile Glu Leu His Asn Gly
50 55 60
Glu Tyr Ser Gln Gly Lys His Gly Phe Thr Met Ala Met Asn Ala Phe
65 70 75 80
Gly Asp Met Thr Asn Glu Glu Phe Arg Gln Met Met Gly Cys Phe Arg
85 90 95
Asn Gln Lys Phe Arg Lys Gly Lys Val Phe Arg Glu Pro Leu Phe Leu
100 105 110
Asp Leu Pro Lys Ser Val Asp Trp Arg Lys Lys Gly Tyr Val Thr Pro
115 120 125
Val Lys Asn Gln Lys Gln Cys Gly Ser Cys Trp Ala Phe Ser Ala Thr
130 135 140
Gly Ala Leu Glu Gly Gln Met Phe Arg Lys Thr Gly Lys Leu Val Ser
145 150 155 160
Leu Ser Glu Gln Asn Leu Val Asp Cys Ser Arg Pro Gln Gly Asn Gln
165 170 175
Gly Cys Asn Gly Gly Phe Met Ala Arg Ala Phe Gln Tyr Val Lys Glu
180 185 190
Asn Gly Gly Leu Asp Ser Glu Glu Ser Tyr Pro TyrVal Ala Val Asp
195 200 205
Glu Ile Cys Lys Tyr Arg Pro Glu Asn Ser Val Ala Asn Asp Thr Gly
210 215 220
Phe Thr Val Val Ala Pro Gly Lys Glu Lys Ala Leu Met Lys Ala Val
225 230 235 240
Ala Thr Val Gly Pro Ile Ser Val Ala Met Asp Ala Gly His Ser Ser
245 250 255
Phe Gln Phe Tyr Lys Ser Gly Ile Tyr Phe Glu Pro Asp Cys Ser Ser
260 265 270
Lys Asn Leu Asp His Gly Val Leu Val Val Gly Tyr Gly Phe Glu Gly
275 280 285
Ala Asn Ser Asn Asn Ser Lys Tyr Trp Leu Val Lys Asn Ser Trp Gly
290 295 300
Pro Glu Trp Gly Ser Asn Gly Tyr Val Lys Ile Ala Lys Asp Lys Asn
305 310 315 320
Asn His Cys Gly Ile Ala Thr Ala Ala Ser Tyr Pro Asn Val
325 330
<210>43
<211>321
<212>PRT
<213> human (Homo sapiens)
<400>43
Met Asp Val Arg Ala Leu Pro Trp Leu Pro Trp Leu Leu Trp Leu Leu
1 5 10 15
Cys Arg Gly Gly Gly Asp Ala Asp Ser Arg Ala Pro Phe Thr Pro Thr
20 25 30
Trp Pro Arg Ser Arg Glu Arg Glu Ala Ala Ala Phe Arg Glu Ser Leu
35 40 45
Asn Arg His Arg Tyr Leu Asn Ser Leu Phe Pro Ser Glu Asn Ser Thr
50 55 60
Ala Phe Tyr Gly Ile Asn Gln Phe Ser Tyr Leu Phe Pro Glu Glu Phe
65 70 75 80
Lys Ala Ile Tyr Leu Arg Ser Lys Pro Ser Lys Phe Pro Arg Tyr Ser
85 90 95
Ala Glu Val His Met Ser Ile Pro Asn Val Ser Leu Pro Leu Arg Phe
100 105 110
Asp Trp Arg Asp Lys Gln Val Val Thr Gln Val ArgAsn Gln Gln Met
115 120 125
Cys Gly Gly Cys Trp Ala Phe Ser Val Val Gly Ala Val Glu Ser Ala
130 135 140
Tyr Ala Ile Lys Gly Lys Pro Leu Glu Asp Leu Ser Val Gln Gln Val
145 150 155 160
Ile Asp Cys Ser Tyr Asn Asn Tyr Gly Cys Asn Gly Gly Ser Thr Leu
165 170 175
Asn Ala Leu Asn Trp Leu Asn Lys Met Gln Val Lys Leu Val Lys Asp
180 185 190
Ser Glu Tyr Pro Phe Lys Ala Gln Asn Gly Leu Cys His Tyr Phe Ser
195 200 205
Gly Ser His Ser Gly Phe Ser Ile Lys Gly Tyr Ser Ala Tyr Asp Phe
210 215 220
Ser Asp Gln Glu Asp Glu Met Ala Lys Ala Leu Leu Thr Phe Gly Pro
225 230 235 240
Leu Val Val Ile Val Asp Ala Val Ser Trp Gln Asp Tyr Leu Gly Gly
245 250 255
Ile Ile Gln His His Cys Ser Ser Gly Glu Ala Asn His Ala Val Leu
260 265 270
Ile Thr Gly Phe Asp Lys Thr Gly Ser Thr Pro Tyr Trp Ile Val Arg
275 280 285
Asn Ser Trp Gly Ser Ser Trp Gly Val Asp Gly Tyr Ala His Val Lys
290 295 300
Met Gly Ser Asn Val Cys Gly Ile Ala Asp Ser Val Ser Ser Ile Phe
305 310 315 320
Val
<210>44
<211>331
<212>PRT
<213> human (Homo sapiens)
<400>44
Met Lys Arg Leu Val Cys Val Leu Leu Val Cys Ser Ser Ala Val Ala
1 5 10 15
Gln Leu His Lys Asp Pro Thr Leu Asp His His Trp His Leu Trp Lys
20 25 30
Lys Thr Tyr Gly Lys Gln Tyr Lys Glu Lys Asn Glu Glu Ala Val Arg
35 40 45
Arg Leu Ile Trp Glu Lys Asn Leu Lys Phe Val Met Leu His Asn Leu
50 55 60
Glu His Ser Met Gly Met His Ser Tyr Asp Leu Gly Met Asn His Leu
65 70 75 80
Gly Asp Met Thr Ser Glu Glu Val Met Ser Leu Met Ser Ser Leu Arg
85 90 95
Val Pro Ser Gln Trp Gln Arg Asn Ile Thr Tyr Lys Ser Asn Pro Asn
100 105 110
Arg Ile Leu Pro Asp Ser Val Asp Trp Arg Glu Lys Gly Cys Val Thr
115 120 125
Glu Val Lys Tyr Gln Gly Ser Cys Gly Ala Cys Trp Ala Phe Ser Ala
130 135 140
Val Gly Ala Leu Glu Ala Gln Leu Lys Leu Lys Thr Gly Lys Leu Val
145 150 155 160
Ser Leu Ser Ala Gln Asn Leu Val Asp Cys Ser Thr Glu Lys Tyr Gly
165 170 175
Asn Lys Gly Cys Asn Gly Gly Phe Met Thr Thr Ala Phe Gln Tyr Ile
180185 190
Ile Asp Asn Lys Gly Ile Asp Ser Asp Ala Ser Tyr Pro Tyr Lys Ala
195 200 205
Met Asp Gln Lys Cys Gln Tyr Asp Ser Lys Tyr Arg Ala Ala Thr Cys
210 215 220
Ser Lys Tyr Thr Glu Leu Pro Tyr Gly Arg Glu Asp Val Leu Lys Glu
225 230 235 240
Ala Val Ala Asn Lys Gly Pro Val Ser Val Gly Val Asp Ala Arg His
245 250 255
Pro Ser Phe Phe Leu Tyr Arg Ser Gly Val Tyr Tyr Glu Pro Ser Cys
260 265 270
Thr Gln Asn Val Asn His Gly Val Leu Val Val Gly Tyr Gly Asp Leu
275 280 285
Asn Gly Lys Glu Tyr Trp Leu Val Lys Asn Ser Trp Gly His Asn Phe
290 295 300
Gly Glu Glu Gly Tyr Ile Arg Met Ala Arg Asn Lys Gly Asn His Cys
305 310 315 320
Gly Ile Ala Ser Phe Pro Ser Tyr Pro Glu Ile
325 330
<210>45
<211>376
<212>PRT
<213> human (Homo sapiens)
<400>45
Met Ala Leu Thr Ala His Pro Ser Cys Leu Leu Ala Leu Leu Val Ala
1 5 10 15
Gly Leu Ala Gln Gly Ile Arg Gly Pro Leu Arg Ala Gln Asp Leu Gly
20 25 30
Pro Gln Pro Leu Glu Leu Lys Glu Ala Phe Lys Leu Phe Gln Ile Gln
35 40 45
Phe Asn Arg Ser Tyr Leu Ser Pro Glu Glu His Ala His Arg Leu Asp
50 55 60
Ile Phe Ala His Asn Leu Ala Gln Ala Gln Arg Leu Gln Glu Glu Asp
65 70 75 80
Leu Gly Thr Ala Glu Phe Gly Val Thr Pro Phe Ser Asp Leu Thr Glu
85 90 95
Glu Glu Phe Gly Gln Leu Tyr Gly Tyr Arg Arg Ala Ala Gly Gly Val
100105 110
Pro Ser Met Gly Arg Glu Ile Arg Ser Glu Glu Pro Glu Glu Ser Val
115 120 125
Pro Phe Ser Cys Asp Trp Arg Lys Val Ala Ser Ala Ile Ser Pro Ile
130 135 140
Lys Asp Gln Lys Asn Cys Asn Cys Cys Trp Ala Met Ala Ala Ala Gly
145 150 155 160
Asn Ile Glu Thr Leu Trp Arg Ile Ser Phe Trp Asp Phe Val Asp Val
165 170 175
Ser Val Gln Glu Leu Leu Asp Cys Gly Arg Cys Gly Asp Gly Cys His
180 185 190
Gly Gly Phe Val Trp Asp Ala Phe Ile Thr Val Leu Asn Asn Ser Gly
195 200 205
Leu Ala Ser Glu Lys Asp Tyr Pro Phe Gln Gly Lys Val Arg Ala His
210 215 220
Arg Cys His Pro Lys Lys Tyr Gln Lys Val Ala Trp Ile Gln Asp Phe
225 230 235 240
Ile Met Leu Gln Asn Asn Glu His Arg Ile Ala Gln Tyr Leu Ala Thr
245 250 255
Tyr Gly Pro Ile Thr Val Thr Ile Asn Met Lys Pro Leu Gln Leu Tyr
260 265 270
Arg Lys Gly Val Ile Lys Ala Thr Pro Thr Thr Cys Asp Pro Gln Leu
275 280 285
Val Asp His Ser Val Leu Leu Val Gly Phe Gly Ser Val Lys Ser Glu
290 295 300
Glu Gly Ile Trp Ala Glu Thr Val Ser Ser Gln Ser Gln Pro Gln Pro
305 310 315 320
Pro His Pro Thr Pro Tyr Trp Ile Leu Lys Asn Ser Trp Gly Ala Gln
325 330 335
Trp Gly Glu Lys Gly Tyr Phe Arg Leu His Arg Gly Ser Asn Thr Cys
340 345 350
Gly Ile Thr Lys Phe Pro Leu Thr Ala Arg Val Gln Lys Pro Asp Met
355 360 365
Lys Pro Arg Val Ser Cys Pro Pro
370 375
<210>46
<211>303
<212>PRT
<213> human (Homo sapiens)
<400>46
Met Ala Arg Arg Gly Pro Gly Trp Arg Pro Leu Leu Leu Leu Val Leu
1 5 10 15
Leu Ala Gly Ala Ala Gln Gly Gly Leu Tyr Phe Arg Arg Gly Gln Thr
20 25 30
Cys Tyr Arg Pro Leu Arg Gly Asp Gly Leu Ala Pro Leu Gly Arg Ser
35 40 45
Thr Tyr Pro Arg Pro His Glu Tyr Leu Ser Pro Ala Asp Leu Pro Lys
50 55 60
Ser Trp Asp Trp Arg Asn Val Asp Gly Val Asn Tyr Ala Ser Ile Thr
65 70 75 80
Arg Asn Gln His Ile Pro Gln Tyr Cys Gly Ser Cys Trp Ala His Ala
85 90 95
Ser Thr Ser Ala Met Ala Asp Arg Ile Asn Ile Lys Arg Lys Gly Ala
100 105 110
Trp Pro Ser Thr Leu Leu Ser Val Gln Asn Val Ile Asp Cys Gly Asn
115 120 125
Ala Gly Ser Cys Glu Gly Gly Asn Asp Leu Ser Val Trp Asp Tyr Ala
130 135 140
His Gln His Gly Ile Pro Asp Glu Thr Cys Asn Asn Tyr Gln Ala Lys
145 150 155 160
Asp Gln Glu Cys Asp Lys Phe Asn Gln Cys Gly Thr Cys Asn Glu Phe
165 170 175
Lys Glu Cys His Ala Ile Arg Asn Tyr Thr Leu Trp Arg Val Gly Asp
180 185 190
Tyr Gly Ser Leu Ser Gly Arg Glu Lys Met Met Ala Glu Ile Tyr Ala
195 200 205
Asn Gly Pro Ile Ser Cys Gly Ile Met Ala Thr Glu Arg Leu Ala Asn
210 215 220
Tyr Thr Gly Gly Ile Tyr Ala Glu Tyr Gln Asp Thr Thr Tyr Ile Asn
225 230 235 240
His Val Val Ser Val Ala Gly Trp Gly Ile Ser Asp Gly Thr Glu Tyr
245 250 255
Trp Ile Val Arg Asn Ser Trp Gly Glu Pro Trp Gly Glu Arg Gly Trp
260 265 270
Leu Arg Ile Val Thr Ser Thr Tyr Lys Asp Gly Lys Gly Ala Arg Tyr
275 280 285
Asn Leu Ala Ile Glu Glu His Cys Thr Phe Gly Asp Pro Ile Val
290 295 300
<210>47
<211>338
<212>PRT
<213> human (Homo sapiens)
<400>47
Met Leu Gln Lys Pro Lys Ser Val Lys Leu Arg Ala Leu Arg Ser Pro
1 5 10 15
Arg Lys Phe Gly Val Ala Gly Arg Ser Cys Gln Glu Val Leu Arg Lys
20 25 30
Gly Cys Leu Arg Phe Gln Leu Pro Glu Arg Gly Ser Arg Leu Cys Leu
35 40 45
Tyr Glu Asp Gly Thr Glu Leu Thr Glu Asp Tyr Phe Pro Ser Val Pro
50 55 60
Asp Asn Ala Glu Leu Val Leu Leu Thr Leu Gly Gln Ala Trp Gln Gly
65 70 75 80
Tyr Val Ser Asp Ile Arg Arg Phe Leu Ser Ala Phe His Glu Pro Gln
85 90 95
Val Gly Leu Ile Gln Ala Ala Gln Gln Leu Leu Cys Asp Glu Gln Ala
100 105 110
Pro Gln Arg Gln Arg Leu Leu Ala Asp Leu Leu His Asn Val Ser Gln
115 120 125
Asn Ile Ala Ala Glu Thr Arg Ala Glu Asp Pro Pro Trp Phe Glu Gly
130 135 140
Leu Glu Ser Arg Phe Gln Ser Lys Ser Gly Tyr Leu Arg Tyr Ser Cys
145 150 155 160
Glu Ser Arg Ile Arg Ser Tyr Leu Arg Glu Val Ser Ser Tyr Pro Ser
165 170 175
Thr Val Gly Ala Glu Ala Gln Glu Glu Phe Leu Arg Val Leu Gly Ser
180 185 190
Met Cys Gln Arg Leu Arg Ser Met Gln Tyr Asn Gly Ser Tyr Phe Asp
195 200 205
Arg Gly Ala Lys Gly Gly Ser Arg Leu Cys Thr Pro Glu Gly Trp Phe
210 215 220
Ser Cys Gln Gly Pro Phe Asp Met Asp Ser Cys Leu Ser Arg His Ser
225 230 235 240
Ile Asn Pro Tyr Ser Asn Arg Glu Ser Arg Ile Leu Phe Ser Thr Trp
245 250 255
Asn Leu Asp His Ile Ile Glu Lys Lys Arg Thr Ile Ile Pro Thr Leu
260 265 270
Val Glu Ala Ile Lys Glu Gln Asp Gly Arg Glu Val Asp Trp Glu Tyr
275 280 285
Phe Tyr Gly Leu Leu Phe Thr Ser Glu Asn Leu Lys Leu Val His Ile
290 295 300
Val Cys His Lys Lys Thr Thr His Lys Leu Asn Cys Asp Pro Ser Arg
305 310 315 320
Ile Tyr Lys Pro Gln Thr Arg Leu Lys Arg Lys Gln Pro Val Arg Lys
325 330 335
Arg Gln
<210>48
<211>360
<212>PRT
<213> human (Homo sapiens)
<400>48
Met Ser Gln Glu Arg Pro Thr Phe Tyr Arg Gln Glu Leu Asn Lys Thr
1 5 10 15
Ile Trp Glu Val Pro Glu Arg Tyr Gln Asn Leu Ser Pro Val Gly Ser
20 25 30
Gly Ala Tyr Gly Ser Val Cys Ala Ala Phe Asp Thr Lys Thr Gly Leu
35 40 45
Arg Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Ile Ile His
50 55 60
Ala Lys Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Met Lys His
65 70 75 80
Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Arg Ser Leu
85 90 95
Glu Glu Phe Asn Asp Val Tyr Leu Val Thr His Leu Met Gly Ala Asp
100 105 110
Leu Asn Asn Ile Val Lys Cys Gln Lys Leu Thr Asp Asp His Val Gln
115 120 125
Phe Leu Ile Tyr Gln Ile Leu Arg Gly Leu Lys Tyr Ile His Ser Ala
130 135 140
Asp Ile Ile His Arg Asp Leu Lys Pro Ser Asn Leu Ala Val Asn Glu
145 150 155 160
Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Thr Asp
165 170 175
Asp Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu
180 185 190
Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser
195 200 205
Val Gly Cys Ile Met Ala Glu Leu Leu Thr Gly Arg Thr Leu Phe Pro
210 215 220
Gly Thr Asp His Ile Asp Gln Leu Lys Leu Ile Leu Arg Leu Val Gly
225 230 235 240
Thr Pro Gly Ala Glu Leu Leu Lys Lys Ile Ser Ser Glu Ser Ala Arg
245 250 255
Asn Tyr Ile Gln Ser Leu Thr Gln Met Pro Lys Met Asn Phe Ala Asn
260 265 270
Val Phe Ile Gly Ala Asn Pro Leu Ala Val Asp Leu Leu Glu Lys Met
275 280 285
Leu Val Leu Asp Ser Asp Lys Arg Ile Thr Ala Ala Gln Ala Leu Ala
290 295 300
His Ala Tyr Phe Ala Gln Tyr His Asp Pro Asp Asp Glu Pro Val Ala
305 310 315 320
Asp Pro Tyr Asp Gln Ser Phe Glu Ser Arg Asp Leu Leu Ile Asp Glu
325 330 335
Trp Lys Ser Leu Thr Tyr Asp Glu Val Ile Ser Phe Val Pro Pro Pro
340 345 350
Leu Asp Gln Glu Glu Met Glu Ser
355 360
<210>49
<211>364
<212>PRT
<213> human (Homo sapiens)
<400>49
Met Ser Gly Pro Arg Ala Gly Phe Tyr Arg Gln Glu Leu Asn Lys Thr
1 5 10 15
Val Trp Glu Val Pro Gln Arg Leu Gln Gly Leu Arg Pro Val Gly Ser
20 25 30
Gly Ala Tyr Gly Ser Val Cys Ser Ala Tyr Asp Ala Arg Leu Arg Gln
35 40 45
Lys Val Ala Val Lys Lys Leu Ser Arg Pro Phe Gln Ser Leu Ile His
50 55 60
Ala Arg Arg Thr Tyr Arg Glu Leu Arg Leu Leu Lys His Leu Lys His
65 70 75 80
Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Thr Ser Ile
85 90 95
Glu Asp Phe Ser Glu Val Tyr Leu Val Thr Thr Leu Met Gly Ala Asp
100 105 110
Leu Asn Asn Ile Val Lys Cys Gln Ala Leu Ser Asp Glu His Val Gln
115 120 125
Phe Leu Val Tyr Gln Leu Leu Arg Gly Leu Lys Tyr Ile His Ser Ala
130 135 140
Gly Ile Ile His Arg Asp Leu Lys Pro Ser Asn Val Ala Val Asn Glu
145 150 155160
Asp Cys Glu Leu Arg Ile Leu Asp Phe Gly Leu Ala Arg Gln Ala Asp
165 170 175
Glu Glu Met Thr Gly Tyr Val Ala Thr Arg Trp Tyr Arg Ala Pro Glu
180 185 190
Ile Met Leu Asn Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser
195 200 205
Val Gly Cys Ile Met Ala Glu Leu Leu Gln Gly Lys Ala Leu Phe Pro
210 215 220
Gly Ser Asp Tyr Ile Asp Gln Leu Lys Arg Ile Met Glu Val Val Gly
225 230 235 240
Thr Pro Ser Pro Glu Val Leu Ala Lys Ile Ser Ser Glu His Ala Arg
245 250 255
Thr Tyr Ile Gln Ser Leu Pro Pro Met Pro Gln Lys Asp Leu Ser Ser
260 265 270
Ile Phe Arg Gly Ala Asn Pro Leu Ala Ile Asp Leu Leu Gly Arg Met
275 280 285
Leu Val Leu Asp Ser Asp Gln Arg Val Ser Ala Ala Glu Ala Leu Ala
290 295 300
His Ala Tyr Phe Ser Gln Tyr His Asp Pro Glu Asp Glu Pro Glu Ala
305 310 315 320
Glu Pro Tyr Asp Glu Ser Val Glu Ala Lys Glu Arg Thr Leu Glu Glu
325 330 335
Trp Lys Glu Leu Thr Tyr Gln Glu Val Leu Ser Phe Lys Pro Pro Glu
340 345 350
Pro Pro Lys Pro Pro Gly Ser Leu Glu Ile Glu Gln
355 360
<210>50
<211>367
<212>PRT
<213> human (Homo sapiens)
<400>50
Met Ser Ser Pro Pro Pro Ala Arg Ser Gly Phe Tyr Arg Gln Glu Val
1 5 10 15
Thr Lys Thr Ala Trp Glu Val Arg Ala Val Tyr Arg Asp Leu Gln Pro
20 25 30
Val Gly Ser Gly Ala Tyr Gly Ala Val Cys Ser Ala Val Asp Gly Arg
35 40 45
Thr Gly Ala Lys Val Ala Ile Lys Lys Leu Tyr Arg Pro Phe Gln Ser
50 55 60
Glu Leu Phe Ala Lys Arg Ala Tyr Arg Glu Leu Arg Leu Leu Lys His
65 70 75 80
Met Arg His Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Asp
85 90 95
Glu Thr Leu Asp Asp Phe Thr Asp Phe Tyr Leu Val Met Pro Phe Met
100 105 110
Gly Thr Asp Leu Gly Lys Leu Met Lys His Glu Lys Leu Gly Glu Asp
115 120 125
Arg Ile Gln Phe Leu Val Tyr Gln Met Leu Lys Gly Leu Arg Tyr Ile
130 135 140
His Ala Ala Gly Ile Ile His Arg Asp Leu Lys Pro Gly Asn Leu Ala
145 150 155 160
Val Asn Glu Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg
165 170 175
Gln Ala Asp Ser Glu Met Thr Gly Tyr Val Val Thr Arg Trp Tyr Arg
180 185 190
Ala Pro Glu Val Ile Leu Asn Trp Met Arg Tyr Thr Gln Thr Val Asp
195 200 205
Ile Trp Ser Val Gly Cys Ile Met Ala Glu Met Ile Thr Gly Lys Thr
210 215 220
Leu Phe Lys Gly Ser Asp His Leu Asp Gln Leu Lys Glu Ile Met Lys
225 230 235 240
Val Thr Gly Thr Pro Pro Ala Glu Phe Val Gln Arg Leu Gln Ser Asp
245 250 255
Glu Ala Lys Asn Tyr Met Lys Gly Leu Pro Glu Leu Glu Lys Lys Asp
260 265 270
Phe Ala Ser Ile Leu Thr Asn Ala Ser Pro Leu Ala Val Asn Leu Leu
275 280 285
Glu Lys Met Leu Val Leu Asp Ala Glu Gln Arg Val Thr Ala Gly Glu
290 295 300
Ala Leu Ala His Pro Tyr Phe Glu Ser Leu His Asp Thr Glu Asp Glu
305 310 315 320
Pro Gln Val Gln Lys Tyr Asp Asp Ser Phe Asp Asp Val Asp Arg Thr
325 330 335
Leu Asp Glu Trp Lys Arg Val Thr Tyr Lys Glu Val Leu Ser Phe Lys
340 345 350
Pro Pro Arg Gln Leu Gly Ala Arg Val Ser Lys Glu Thr Pro Leu
355 360 365
<210>51
<211>365
<212>PRT
<213> human (Homo sapiens)
<400>51
Met Ser Leu Ile Arg Lys Lys Gly Phe Tyr Lys Gln Asp Val Asn Lys
1 5 10 15
Thr Ala Trp Glu Leu Pro Lys Thr Tyr Val Ser Pro Thr His Val Gly
20 25 30
Ser Gly Ala Tyr Gly Ser Val Cys Ser Ala Ile Asp Lys Arg Ser Gly
35 40 45
Glu Lys Val Ala Ile Lys Lys Leu Ser Arg Pro Phe Gln Ser Glu Ile
50 55 60
Phe Ala Lys Arg Ala Tyr Arg Glu Leu Leu Leu Leu Lys His Met Gln
65 70 75 80
His Glu Asn Val Ile Gly Leu Leu Asp Val Phe Thr Pro Ala Ser Ser
85 90 95
Leu Arg Asn Phe Tyr Asp Phe Tyr Leu Val Met Pro Phe Met Gln Thr
100 105 110
Asp Leu Gln Lys Ile Met Gly Met Glu Phe Ser Glu Glu Lys Ile Gln
115 120 125
Tyr Leu Val Tyr Gln Met Leu Lys Gly Leu Lys Tyr Ile His Ser Ala
130 135 140
Gly Val Val His Arg Asp Leu Lys Pro Gly Asn Leu Ala Val Asn Glu
145 150 155 160
Asp Cys Glu Leu Lys Ile Leu Asp Phe Gly Leu Ala Arg His Ala Asp
165 170 175
Ala Glu Met Thr Gly Tyr Val Val Thr Arg Trp Tyr Arg Ala Pro Glu
180 185 190
Val Ile Leu Ser Trp Met His Tyr Asn Gln Thr Val Asp Ile Trp Ser
195 200 205
Val Gly Cys Ile Met Ala Glu Met Leu Thr Gly Lys Thr Leu Phe Lys
210 215 220
Gly Lys Asp Tyr Leu Asp Gln Leu Thr Gln Ile Leu Lys Val Thr Gly
225 230 235 240
Val Pro Gly Thr Glu Phe Val Gln Lys Leu Asn Asp Lys Ala Ala Lys
245 250 255
Ser Tyr Ile Gln Ser Leu Pro Gln Thr Pro Arg Lys Asp Phe Thr Gln
260 265 270
Leu Phe Pro Arg Ala Ser Pro Gln Ala Ala Asp Leu Leu Glu Lys Met
275 280 285
Leu Glu Leu Asp Val Asp Lys Arg Leu Thr Ala Ala Gln Ala Leu Thr
290 295 300
His Pro Phe Phe Glu Pro Phe Arg Asp Pro Glu Glu Glu Thr Glu Ala
305 310 315 320
Gln Gln Pro Phe Asp Asp Ser Leu Glu His Glu Lys Leu Thr Val Asp
325 330 335
Glu Trp Lys Gln His Ile Tyr Lys Glu Ile Val Asn Phe Ser Pro Ile
340 345 350
Ala Arg Lys Asp Ser Arg Arg Arg Ser Gly Met Lys Leu
355 360 365
<210>52
<211>403
<212>PRT
<213> human (Homo sapiens)
<400>52
Met Thr Ala Ile Ile Lys Glu Ile Val Ser Arg Asn Lys Arg Arg Tyr
1 5 10 15
Gln Glu Asp Gly Phe Asp Leu Asp Leu Thr Tyr Ile Tyr Pro Asn Ile
20 25 30
Ile Ala Met Gly Phe Pro Ala Glu Arg Leu Glu Gly Val Tyr Arg Asn
35 40 45
Asn Ile Asp Asp Val Val Arg Phe Leu Asp Ser Lys His Lys Asn His
50 55 60
Tyr Lys Ile Tyr Asn Leu Cys Ala Glu Arg His Tyr Asp Thr Ala Lys
65 70 75 80
Phe Asn Cys Arg Val Ala Gln Tyr Pro Phe Glu Asp His Asn Pro Pro
85 90 95
Gln Leu Glu Leu Ile Lys Pro Phe Cys Glu Asp Leu Asp Gln Trp Leu
100 105 110
Ser Glu Asp Asp Asn His Val Ala Ala Ile His Cys Lys Ala Gly Lys
115 120 125
Gly Arg Thr Gly Val Met Ile Cys Ala Tyr Leu Leu His Arg Gly Lys
130 135 140
Phe Leu Lys Ala Gln Glu Ala Leu Asp Phe Tyr Gly Glu Val Arg Thr
145 150 155 160
Arg Asp Lys Lys Gly Val Thr Ile Pro Ser Gln Arg Arg Tyr Val Tyr
165 170 175
Tyr Tyr Ser Tyr Leu Leu Lys Asn His Leu Asp Tyr Arg Pro Val Ala
180 185 190
Leu Leu Phe His Lys Met Met Phe Glu Thr Ile Pro Met Phe Ser Gly
195 200 205
Gly Thr Cys Asn Pro Gln Phe Val Val Cys Gln Leu Lys Val Lys Ile
210 215 220
Tyr Ser Ser Asn Ser Gly Pro Thr Arg Arg Glu Asp Lys Phe Met Tyr
225 230 235 240
Phe Glu Phe Pro Gln Pro Leu Pro Val Cys Gly Asp Ile Lys Val Glu
245 250 255
Phe Phe His Lys Gln Asn Lys Met Leu Lys Lys Asp Lys Met Phe His
260 265 270
Phe Trp Val Asn Thr Phe Phe Ile Pro Gly Pro Glu Glu Thr Ser Glu
275 280 285
Lys Val Glu Asn Gly Ser Leu Cys Asp Gln Glu Ile Asp Ser Ile Cys
290 295 300
Ser Ile Glu Arg Ala Asp Asn Asp Lys Glu Tyr Leu Val Leu Thr Leu
305 310 315 320
Thr Lys Asn Asp Leu Asp Lys Ala Asn Lys Asp Lys Ala Asn Arg Tyr
325 330 335
Phe Ser Pro Asn Phe Lys Val Lys Leu Tyr Phe Thr Lys Thr Val Glu
340 345 350
Glu Pro Ser Asn Pro Glu Ala Ser Ser Ser Thr Ser Val Thr Pro Asp
355 360 365
Val Ser Asp Asn Glu Pro Asp His Tyr Arg Tyr Ser Asp Thr Thr Asp
370375 380
Ser Asp Pro Glu Asn Glu Pro Phe Asp Glu Asp Gln His Thr Gln Ile
385 390 395 400
Thr Lys Val
<210>53
<211>1154
<212>PRT
<213> human (Homo sapiens)
<400>53
Met Gln Tyr Leu Asn Ile Lys Glu Asp Cys Asn Ala Met Ala Phe Cys
1 5 10 15
Ala Lys Met Arg Ser Ser Lys Lys Thr Glu Val Asn Leu Glu Ala Pro
20 25 30
Glu Pro Gly Val Glu Val Ile Phe Tyr Leu Ser Asp Arg Glu Pro Leu
35 40 45
Arg Leu Gly Ser Gly Glu Tyr Thr Ala Glu Glu Leu Cys Ile Arg Ala
50 55 60
Ala Gln Ala Cys Arg Ile Ser Pro Leu Cys His Asn Leu Phe Ala Leu
65 70 75 80
Tyr Asp Glu Asn Thr Lys Leu Trp Tyr Ala Pro Asn Arg Thr Ile Thr
85 90 95
Val Asp Asp Lys Met Ser Leu Arg Leu His Tyr Arg Met Arg Phe Tyr
100 105 110
Phe Thr Asn Trp His Gly Thr Asn Asp Asn Glu Gln Ser Val Trp Arg
115 120 125
His Ser Pro Lys Lys Gln Lys Asn Gly Tyr Glu Lys Lys Lys Ile Pro
130 135 140
Asp Ala Thr Pro Leu Leu Asp Ala Ser Ser Leu Glu Tyr Leu Phe Ala
145 150 155 160
Gln Gly Gln Tyr Asp Leu Val Lys Cys Leu Ala Pro Ile Arg Asp Pro
165 170 175
Lys Thr Glu Gln Asp Gly His Asp Ile Glu Asn Glu Cys Leu Gly Met
180 185 190
Ala Val Leu Ala Ile Ser His Tyr Ala Met Met Lys Lys Met Gln Leu
195 200 205
Pro Glu Leu Pro Lys Asp Ile Ser Tyr Lys Arg Tyr Ile Pro Glu Thr
210 215 220
Leu Asn Lys Ser Ile Arg Gln Arg Asn Leu Leu Thr Arg MetArg Ile
225 230 235 240
Asn Asn Val Phe Lys Asp Phe Leu Lys Glu Phe Asn Asn Lys Thr Ile
245 250 255
Cys Asp Ser Ser Val Ser Thr His Asp Leu Lys Val Lys Tyr Leu Ala
260 265 270
Thr Leu Glu Thr Leu Thr Lys His Tyr Gly Ala Glu Ile Phe Glu Thr
275 280 285
Ser Met Leu Leu Ile Ser Ser Glu Asn Glu Met Asn Trp Phe His Ser
290 295 300
Asn Asp Gly Gly Asn Val Leu Tyr Tyr Glu Val Met Val Thr Gly Asn
305 310 315 320
Leu Gly Ile Gln Trp Arg His Lys Pro Asn Val Val Ser Val Glu Lys
325 330 335
Glu Lys Asn Lys Leu Lys Arg Lys Lys Leu Glu Asn Lys His Lys Lys
340 345 350
Asp Glu Glu Lys Asn Lys Ile Arg Glu Glu Trp Asn Asn Phe Ser Tyr
355 360 365
Phe Pro Glu Ile Thr His Ile Val Ile Lys Glu Ser Val Val Ser Ile
370 375 380
Asn Lys Gln Asp Asn Lys Lys Met Glu Leu Lys Leu Ser Ser His Glu
385 390 395 400
Glu Ala Leu Ser Phe Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr
405 410 415
Ala Asp Ala His His Tyr Leu Cys Thr Asp Val Ala Pro Pro Leu Ile
420 425 430
Val His Asn Ile Gln Asn Gly Cys His Gly Pro Ile Cys Thr Glu Tyr
435 440 445
Ala Ile Asn Lys Leu Arg Gln Glu Gly Ser Glu Glu Gly Met Tyr Val
450 455 460
Leu Arg Trp Ser Cys Thr Asp Phe Asp Asn Ile Leu Met Thr Val Thr
465 470 475 480
Cys Phe Glu Lys Ser Glu Gln Val Gln Gly Ala Gln Lys Gln Phe Lys
485 490 495
Asn Phe Gln Ile Glu Val Gln Lys Gly Arg Tyr Ser Leu His Gly Ser
500 505510
Asp Arg Ser Phe Pro Ser Leu Gly Asp Leu Met Ser His Leu Lys Lys
515 520 525
Gln Ile Leu Arg Thr Asp Asn Ile Ser Phe Met Leu Lys Arg Cys Cys
530 535 540
Gln Pro Lys Pro Arg Glu Ile Ser Asn Leu Leu Val Ala Thr Lys Lys
545 550 555 560
Ala Gln Glu Trp Gln Pro Val Tyr Pro Met Ser Gln Leu Ser Phe Asp
565 570 575
Arg Ile Leu Lys Lys Asp Leu Val Gln Gly Glu His Leu Gly Arg Gly
580 585 590
Thr Arg Thr His Ile Tyr Ser Gly Thr Leu Met Asp Tyr Lys Asp Asp
595 600 605
Glu Gly Thr Ser Glu Glu Lys Lys Ile Lys Val Ile Leu Lys Val Leu
610 615 620
Asp Pro Ser His Arg Asp Ile Ser Leu Ala Phe Phe Glu Ala Ala Ser
625 630 635 640
Met Met Arg Gln Val Ser His Lys His Ile Val Tyr Leu Tyr Gly Val
645 650 655
Cys Val Arg Asp Val Glu Asn Ile Met Val Glu Glu Phe Val Glu Gly
660 665 670
Gly Pro Leu Asp Leu Phe Met His Arg Lys Ser Asp Val Leu Thr Thr
675 680 685
Pro Trp Lys Phe Lys Val Ala Lys Gln Leu Ala Ser Ala Leu Ser Tyr
690 695 700
Leu Glu Asp Lys Asp Leu Val His Gly Asn Val Cys Thr Lys Asn Leu
705 710 715 720
Leu Leu Ala Arg Glu Gly Ile Asp Ser Glu Cys Gly Pro Phe Ile Lys
725 730 735
Leu Ser Asp Pro Gly Ile Pro Ile Thr Val Leu Ser Arg Gln Glu Cys
740 745 750
Ile Glu Arg Ile Pro Trp Ile Ala Pro Glu Cys Val Glu Asp Ser Lys
755 760 765
Asn Leu Ser Val Ala Ala Asp Lys Trp Ser Phe Gly Thr Thr Leu Trp
770 775 780
Glu Ile Cys Tyr Asn Gly Glu Ile Pro Leu Lys Asp Lys Thr Leu Ile
785 790 795 800
Glu Lys Glu Arg Phe Tyr Glu Ser Arg Cys Arg Pro Val Thr Pro Ser
805 810 815
Cys Lys Glu Leu Ala Asp Leu Met Thr Arg Cys Met Asn Tyr Asp Pro
820 825 830
Asn Gln Arg Pro Phe Phe Arg Ala Ile Met Arg Asp Ile Asn Lys Leu
835 840 845
Glu Glu Gln Asn Pro Asp Ile Val Ser Glu Lys Lys Pro Ala Thr Glu
850 855 860
Val Asp Pro Thr His Phe Glu Lys Arg Phe Leu Lys Arg Ile Arg Asp
865 870 875 880
Leu Gly Glu Gly His Phe Gly Lys Val Glu Leu Cys Arg Tyr Asp Pro
885 890 895
Glu Gly Asp Asn Thr Gly Glu Gln Val Ala Val Lys Ser Leu Lys Pro
900 905 910
Glu Ser Gly Gly Asn His Ile Ala Asp Leu Lys Lys Glu Ile Glu Ile
915 920 925
Leu Arg Asn Leu Tyr His Glu Asn Ile Val Lys Tyr Lys Gly Ile Cys
930 935 940
Thr Glu Asp Gly Gly Asn Gly Ile Lys Leu Ile Met Glu Phe Leu Pro
945 950 955 960
Ser Gly Ser Leu Lys Glu Tyr Leu Pro Lys Asn Lys Asn Lys Ile Asn
965 970 975
Leu Lys Gln Gln Leu Lys Tyr Ala Val Gln Ile Cys Lys Gly Met Asp
980 985 990
Tyr Leu Gly Ser Arg Gln Tyr Val His Arg Asp Leu Ala Ala Arg Asn
995 1000 1005
Val Leu Val Glu Ser Glu His Gln Val Lys Ile Gly Asp Phe Gly Leu
1010 1015 1020
Thr Lys Ala Ile Glu Thr Asp Lys Glu Tyr Tyr Thr Val Lys Asp Asp
1025 1030 1035 1040
Arg Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu Cys Leu Met Gln Ser
1045 1050 1055
Lys Phe Tyr Ile Ala Ser Asp Val Trp Ser Phe Gly Val Thr Leu His
1060 10651070
Glu Leu Leu Thr Tyr Cys Asp Ser Asp Ser Ser Pro Met Ala Leu Phe
1075 1080 1085
Leu Lys Met Ile Gly Pro Thr His Gly Gln Met Thr Val Thr Arg Leu
1090 1095 1100
Val Asn Thr Leu Lys Glu Gly Lys Arg Leu Pro Cys Pro Pro Asn Cys
1105 1110 1115 1120
Pro Asp Glu Val Tyr Gln Leu Met Arg Lys Cys Trp Glu Phe Gln Pro
1125 1130 1135
Ser Asn Arg Thr Ser Phe Gln Asn Leu Ile Glu Gly Phe Glu Ala Leu
1140 1145 1150
Leu Lys
<210>54
<211>1132
<212>PRT
<213> human (Homo sapiens)
<400>54
Met Gly Met Ala Cys Leu Thr Met Thr Glu Met Glu Gly Thr Ser Thr
1 5 10 15
Ser Ser Ile Tyr Gln Asn Gly Asp Ile Ser Gly Asn Ala Asn Ser Met
20 25 30
Lys Gln Ile Asp Pro Val Leu Gln Val Tyr Leu Tyr His Ser Leu Gly
35 40 45
Lys Ser Glu Ala Asp Tyr Leu Thr Phe Pro Ser Gly Glu Tyr Val Ala
50 55 60
Glu Glu Ile Cys Ile Ala Ala Ser Lys Ala Cys Gly Ile Thr Pro Val
65 70 75 80
Tyr His Asn Met Phe Ala Leu Met Ser Glu Thr Glu Arg Ile Trp Tyr
85 90 95
Pro Pro Asn His Val Phe His Ile Asp Glu Ser Thr Arg His Asn Val
100 105 110
Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Arg Trp Tyr Cys Ser Gly Ser
115 120 125
Asn Arg Ala Tyr Arg His Gly Ile Ser Arg Gly Ala Glu Ala Pro Leu
130 135 140
Leu Asp Asp Phe Val Met Ser Tyr Leu Phe Ala Gln Trp Arg His Asp
145 150 155 160
Phe Val His Gly Trp Ile Lys Val Pro Val Thr His Glu Thr Gln Glu
165 170 175
Glu Cys Leu Gly Met Ala Val Leu Asp Met Met Arg Ile Ala Lys Glu
180 185 190
Asn Asp Gln Thr Pro Leu Ala Ile Tyr Asn Ser Ile Ser Tyr Lys Thr
195 200 205
Phe Leu Pro Lys Cys Ile Arg Ala Lys Ile Gln Asp Tyr His Ile Leu
210 215 220
Thr Arg Lys Arg Ile Arg Tyr Arg Phe Arg Arg Phe Ile Gln Gln Phe
225 230 235 240
Ser Gln Cys Lys Ala Thr Ala Arg Asn Leu Lys Leu Lys Tyr Leu Ile
245 250 255
Asn Leu Glu Thr Leu Gln Ser Ala Phe Tyr Thr Glu Lys Phe Glu Val
260 265 270
Lys Glu Pro Gly Ser Gly Pro Ser Gly Glu Glu Ile Phe Ala Thr Ile
275 280 285
Ile Ile Thr Gly Asn Gly Gly Ile Gln Trp Ser Arg Gly Lys His Lys
290 295 300
Glu Ser Glu ThrLeu Thr Glu Gln Asp Leu Gln Leu Tyr Cys Asp Phe
305 310 315 320
Pro Asn Ile Ile Asp Val Ser Ile Lys Gln Ala Asn Gln Glu Gly Ser
325 330 335
Asn Glu Ser Arg Val Val Thr Ile His Lys Gln Asp Gly Lys Asn Leu
340 345 350
Glu Ile Glu Leu Ser Ser Leu Arg Glu Ala Leu Ser Phe Val Ser Leu
355 360 365
Ile Asp Gly Tyr Tyr Arg Leu Thr Ala Asp Ala His His Tyr Leu Cys
370 375 380
Lys Glu Val Ala Pro Pro Ala Val Leu Glu Asn Ile Gln Ser Asn Cys
385 390 395 400
His Gly Pro Ile Ser Met Asp Phe Ala Ile Ser Lys Leu Lys Lys Ala
405 410 415
Gly Asn Gln Thr Gly Leu Tyr Val Leu Arg Cys Ser Pro Lys Asp Phe
420 425 430
Asn Lys Tyr Phe Leu Thr Phe Ala Val Glu Arg Glu Asn Val Ile Glu
435 440 445
Tyr Lys His Cys Leu Ile Thr Lys Asn Glu Asn Glu Glu Tyr Asn Leu
450 455 460
Ser Gly Thr Lys Lys Asn Phe Ser Ser Leu Lys Asp Leu Leu Asn Cys
465 470 475 480
Tyr Gln Met Glu Thr Val Arg Ser Asp Asn Ile Ile Phe Gln Phe Thr
485 490 495
Lys Cys Cys Pro Pro Lys Pro Lys Asp Lys Ser Asn Leu Leu Val Phe
500 505 510
Arg Thr Asn Gly Val Ser Asp Val Pro Thr Ser Pro Thr Leu Gln Arg
515 520 525
Pro Thr His Met Asn Gln Met Val Phe His Lys Ile Arg Asn Glu Asp
530 535 540
Leu Ile Phe Asn Glu Ser Leu Gly Gln Gly Thr Phe Thr Lys Ile Phe
545 550 555 560
Lys Gly Val Arg Arg Glu Val Gly Asp Tyr Gly Gln Leu His Glu Thr
565 570 575
Glu Val Leu Leu Lys Val Leu Asp Lys Ala His Arg Asn Tyr Ser Glu
580 585 590
Ser Phe Phe Glu Ala Ala Ser Met Met Ser Lys Leu Ser His Lys His
595 600 605
Leu Val Leu Asn Tyr Gly Val Cys Val Cys Gly Asp Glu Asn Ile Leu
610 615 620
Val Gln Glu Phe Val Lys Phe Gly Ser Leu Asp Thr Tyr Leu Lys Lys
625 630 635 640
Asn Lys Asn Cys Ile Asn Ile Leu Trp Lys Leu Glu Val Ala Lys Gln
645 650 655
Leu Ala Trp Ala Met His Phe Leu Glu Glu Asn Thr Leu Ile His Gly
660 665 670
Asn Val Cys Ala Lys Asn Ile Leu Leu Ile Arg Glu Glu Asp Arg Lys
675 680 685
Thr Gly Asn Pro Pro Phe Ile Lys Leu Ser Asp Pro Gly Ile Ser Ile
690 695 700
Thr Val Leu Pro Lys Asp Ile Leu Gln Glu Arg Ile Pro Trp Val Pro
705 710 715 720
Pro Glu Cys Ile Glu Asn Pro Lys Asn Leu Asn Leu Ala Thr Asp Lys
725 730 735
Trp Ser Phe Gly Thr Thr Leu Trp Glu Ile Cys Ser Gly Gly Asp Lys
740 745 750
Pro Leu Ser Ala Leu Asp Ser Gln Arg Lys Leu Gln Phe Tyr Glu Asp
755 760 765
Arg His Gln Leu Pro Ala Pro Lys Trp Ala Glu Leu Ala Asn Leu Ile
770 775 780
Asn Asn Cys Met Asp Tyr Glu Pro Asp Phe Arg Pro Ser Phe Arg Ala
785 790 795 800
Ile Ile Arg Asp Leu Asn Ser Leu Phe Thr Pro Asp Tyr Glu Leu Leu
805 810 815
Thr Glu Asn Asp Met Leu Pro Asn Met Arg Ile Gly Ala Leu Gly Phe
820 825 830
Ser Gly Ala Phe Glu Asp Arg Asp Pro Thr Gln Phe Glu Glu Arg His
835 840 845
Leu Lys Phe Leu Gln Gln Leu Gly Lys Gly Asn Phe Gly Ser Val Glu
850 855 860
Met Cys ArgTyr Asp Pro Leu Gln Asp Asn Thr Gly Glu Val Val Ala
865 870 875 880
Val Lys Lys Leu Gln His Ser Thr Glu Glu His Leu Arg Asp Phe Glu
885 890 895
Arg Glu Ile Glu Ile Leu Lys Ser Leu Gln His Asp Asn Ile Val Lys
900 905 910
Tyr Lys Gly Val Cys Tyr Ser Ala Gly Arg Arg Asn Leu Lys Leu Ile
915 920 925
Met Glu Tyr Leu Pro Tyr Gly Ser Leu Arg Asp Tyr Leu Gln Lys His
930 935 940
Lys Glu Arg Ile Asp His Ile Lys Leu Leu Gln Tyr Thr Ser Gln Ile
945 950 955 960
Cys Lys Gly Met Glu Tyr Leu Gly Thr Lys Arg Tyr Ile His Arg Asp
965 970 975
Leu Ala Thr Arg Asn Ile Leu Val Glu Asn Glu Asn Arg Val Lys Ile
980 985 990
Gly Asp Phe Gly Leu Thr Lys Val Leu Pro Gln Asp Lys Glu Tyr Tyr
995 1000 1005
Lys Val Lys Glu Pro Gly Glu Ser Pro Ile Phe Trp Tyr Ala Pro Glu
1010 1015 1020
Ser Leu Thr Glu Ser Lys Phe Ser Val Ala Ser Asp Val Trp Ser Phe
1025 1030 1035 1040
Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Ile Glu Lys Ser Lys Ser
1045 1050 1055
Pro Pro Ala Glu Phe Met Arg Met Ile Gly Asn Asp Lys Gln Gly Gln
1060 1065 1070
Met Ile Val Phe His Leu Ile Glu Leu Leu Lys Asn Asn Gly Arg Leu
1075 1080 1085
Pro Arg Pro Asp Gly Cys Pro Asp Glu Ile Tyr Met Ile Met Thr Glu
1090 1095 1100
Cys Trp Asn Asn Asn Val Asn Gln Arg Pro Ser Phe Arg Asp Leu Ala
1105 1110 1115 1120
Leu Arg Val Asp Gln Ile Arg Asp Asn Met Ala Gly
1125 1130
<210>55
<211>1124
<212>PRT
<213> human (Homo sapiens)
<400>55
Met Ala Pro Pro Ser Glu Glu Thr Pro Leu Ile Pro Gln Arg Ser Cys
1 5 10 15
Ser Leu Leu Ser Thr Glu Ala Gly Ala Leu His Val Leu Leu Pro Ala
20 25 30
Arg Gly Pro Gly Pro Pro Gln Arg Leu Ser Phe Ser Phe Gly Asp His
35 40 45
Leu Ala Glu Asp Leu Cys Val Gln Ala Ala Lys Ala Ser Gly Ile Leu
50 55 60
Pro Val Tyr His Ser Leu Phe Ala Leu Ala Thr Glu Asp Leu Ser Cys
65 70 75 80
Trp Phe Pro Pro Ser His Ile Phe Ser Val Glu Asp Ala Ser Thr Gln
85 90 95
Val Leu Leu Tyr Arg Ile Arg Phe Tyr Phe Pro Asn Trp Phe Gly Leu
100 105 110
Glu Lys Cys His Arg Phe Gly Leu Arg Lys Asp Leu Ala Ser Ala Ile
115 120 125
Leu Asp Leu Pro Val Leu Glu His Leu Phe Ala Gln His Arg Ser Asp
130 135 140
Leu Val Ser Gly Arg Leu Pro Val Gly Leu Ser Leu Lys Glu Gln Gly
145 150 155 160
Glu Cys Leu Ser Leu Ala Val Leu Asp Leu Ala Arg Met Ala Arg Glu
165 170 175
Gln Ala Gln Arg Pro Gly Glu Leu Leu Lys Thr Val Ser Tyr Lys Ala
180 185 190
Cys Leu Pro Pro Ser Leu Arg Asp Leu Ile Gln Gly Leu Ser Phe Val
195 200 205
Thr Arg Arg Arg Ile Arg Arg Thr Val Arg Arg Ala Leu Arg Arg Val
210 215 220
Ala Ala Cys Gln Ala Asp Arg His Ser Leu Met Ala Lys Tyr Ile Met
225 230 235 240
Asp Leu Glu Arg Leu Asp Pro Ala Gly Ala Ala Glu Thr Phe His Val
245 250 255
Gly Leu Pro Gly Ala Leu Gly Gly His Asp Gly Leu Gly Leu Leu Arg
260 265 270
Val Ala Gly Asp Gly Gly Ile Ala Trp Thr Gln Gly Glu Gln Glu Val
275 280 285
Leu Gln Pro Phe Cys Asp Phe Pro Glu Ile Val Asp Ile Ser Ile Lys
290 295 300
Gln Ala Pro Arg Val Gly Pro Ala Gly Glu His Arg Leu Val Thr Val
305 310 315 320
Thr Arg Thr Asp Asn Gln Ile Leu Glu Ala Glu Phe Pro Gly Leu Pro
325 330 335
Glu Ala Leu Ser Phe Val Ala Leu Val Asp Gly Tyr Phe Arg Leu Thr
340 345 350
Thr Asp Ser Gln His Phe Phe Cys Lys Glu Val Ala Pro Pro Arg Leu
355 360 365
Leu Glu Glu Val Ala Glu Gln Cys His Gly Pro Ile Thr Leu Asp Phe
370 375 380
Ala Ile Asn Lys Leu Lys Thr Gly Gly Ser Arg Pro Gly Ser Tyr Val
385 390 395 400
Leu Arg Arg Ser Pro Gln Asp Phe Asp Ser Phe Leu Leu Thr Val Cys
405 410 415
Val Gln Asn Pro Leu Gly Pro Asp Tyr Lys Gly Cys Leu Ile Arg Arg
420 425 430
Ser Pro Thr Gly Thr Phe Leu Leu Val Gly Leu Ser Arg Pro His Ser
435 440 445
Ser Leu Arg Glu Leu Leu Ala Thr Cys Trp Asp Gly Gly Leu His Val
450 455 460
Asp Gly Val Ala Val Thr Leu Thr Ser Cys Cys Ile Pro Arg Pro Lys
465 470 475 480
Glu Lys Ser Asn Leu Ile Val Val Gln Arg Gly His Ser Pro Pro Thr
485 490 495
Ser Ser Leu Val Gln Pro Gln Ser Gln Tyr Gln Leu Ser Gln Met Thr
500 505 510
Phe His Lys Ile Pro Ala Asp Ser Leu Glu Trp His Glu Asn Leu Gly
515 520 525
His Gly Ser Phe Thr Lys Ile Tyr Arg Gly Cys Arg His Glu Val Val
530 535 540
Asp Gly GluAla Arg Lys Thr Glu Val Leu Leu Lys Val Met Asp Ala
545 550 555 560
Lys His Lys Asn Cys Met Glu Ser Phe Leu Glu Ala Ala Ser Leu Met
565 570 575
Ser Gln Val Ser Tyr Arg His Leu Val Leu Leu His Gly Val Cys Met
580 585 590
Ala Gly Asp Ser Thr Met Val Gln Glu Phe Val His Leu Gly Ala Ile
595 600 605
Asp Met Tyr Leu Arg Lys Arg Gly His Leu Val Pro Ala Ser Trp Lys
610 615 620
Leu Gln Val Val Lys Gln Leu Ala Tyr Ala Leu Asn Tyr Leu Glu Asp
625 630 635 640
Lys Gly Leu Pro His Gly Asn Val Ser Ala Arg Lys Val Leu Leu Ala
645 650 655
Arg Glu Gly Ala Asp Gly Ser Pro Pro Phe Ile Lys Leu Ser Asp Pro
660 665 670
Gly Val Ser Pro Ala Val Leu Ser Leu Glu Met Leu Thr Asp Arg Ile
675 680 685
Pro Trp Val Ala Pro Glu Cys Leu Arg Glu Ala Gln Thr Leu Ser Leu
690 695 700
Glu Ala Asp Lys Trp Gly Phe Gly Ala Thr Val Trp Glu Val Phe Ser
705 710 715 720
Gly Val Thr Met Pro Ile Ser Ala Leu Asp Pro Ala Lys Lys Leu Gln
725 730 735
Phe Tyr Glu Asp Arg Gln Gln Leu Pro Ala Pro Lys Trp Thr Glu Leu
740 745 750
Ala Leu Leu Ile Gln Gln Cys Met Ala Tyr Glu Pro Val Gln Arg Pro
755 760 765
Ser Phe Arg Ala Val Ile Arg Asp Leu Asn Ser Leu Ile Ser Ser Asp
770 775 780
Tyr Glu Leu Leu Ser Asp Pro Thr Pro Gly Ala Leu Ala Pro Arg Asp
785 790 795 800
Gly Leu Trp Asn Gly Ala Gln Leu Tyr Ala Cys Gln Asp Pro Thr Ile
805 810 815
Phe Glu Glu Arg His Leu Lys Tyr Ile Ser Gln Leu Gly Lys Gly Asn
820 825 830
Phe Gly Ser Val Glu Leu Cys Arg Tyr Asp Pro Leu Gly Asp Asn Thr
835 840 845
Gly Ala Leu Val Ala Val Lys Gln Leu Gln His Ser Gly Pro Asp Gln
850 855 860
Gln Arg Asp Phe Gln Arg Glu Ile Gln Ile Leu Lys Ala Leu His Ser
865 870 875 880
Asp Phe Ile Val Lys Tyr Arg Gly Val Ser Tyr Gly Pro Gly Arg Gln
885 890 895
Ser Leu Arg Leu Val Met Glu Tyr Leu Pro Ser Gly Cys Leu Arg Asp
900 905 910
Phe Leu Gln Arg His Arg Ala Arg Leu Asp Ala Ser Arg Leu Leu Leu
915 920 925
Tyr Ser Ser Gln Ile Cys Lys Gly Met Glu Tyr Leu Gly Ser Arg Arg
930 935 940
Cys Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Glu Ser Glu
945 950 955 960
Ala His Val Lys Ile Ala Asp Phe Gly Leu Ala Lys Leu Leu Pro Leu
965 970 975
Asp Lys Asp Tyr Tyr Val Val Arg Glu Pro Gly Gln Ser Pro Ile Phe
980 985 990
Trp Tyr Ala Pro Glu Ser Leu Ser Asp Asn Ile Phe Ser Arg Gln Ser
995 1000 1005
Asp Val Trp Ser Phe Gly Val Val Leu Tyr Glu Leu Phe Thr Tyr Cys
1010 1015 1020
Asp Lys Ser Cys Ser Pro Ser Ala Glu Phe Leu Arg Met Met Gly Cys
1025 1030 1035 1040
Glu Arg Asp Val Pro Ala Leu Cys Arg Leu Leu Glu Leu Leu Glu Glu
1045 1050 1055
Gly Gln Arg Leu Pro Ala Pro Pro Ala Cys Pro Ala Glu Val His Glu
1060 1065 1070
Leu Met Lys Leu Cys Trp Ala Pro Ser Pro Gln Asp Arg Pro Ser Phe
1075 1080 1085
Ser Ala Leu Gly Pro Gln Leu Asp Met Leu Trp Ser Gly Ser Arg Gly
1090 1095 1100
Cys Glu Thr His Ala Phe Thr Ala His Pro Glu Gly Lys His His Ser
1105 1110 1115 1120
Leu Ser Phe Ser
<210>56
<211>1187
<212>PRT
<213> human (Homo sapiens)
<400>56
Met Pro Leu Arg His Trp Gly Met Ala Arg Gly Ser Lys Pro Val Gly
1 5 10 15
Asp Gly Ala Gln Pro Met Ala Ala Met Gly Gly Leu Lys Val Leu Leu
20 25 30
His Trp Ala Gly Pro Gly Gly Gly Glu Pro Trp Val Thr Phe Ser Glu
35 40 45
Ser Ser Leu Thr Ala Glu Glu Val Cys Ile His Ile Ala His Lys Val
50 55 60
Gly Ile Thr Pro Pro Cys Phe Asn Leu Phe Ala Leu Phe Asp Ala Gln
65 70 75 80
Ala Gln Val Trp Leu Pro Pro Asn His Ile Leu Glu Ile Pro Arg Asp
85 90 95
Ala Ser Leu Met Leu Tyr Phe Arg Ile Arg Phe Tyr Phe Arg Asn Trp
100 105 110
His Gly Met Asn Pro Arg Glu Pro Ala Val Tyr Arg Cys Gly Pro Pro
115 120 125
Gly Thr Glu Ala Ser Ser Asp Gln Thr Ala Gln Gly Met Gln Leu Leu
130 135 140
Asp Pro Ala Ser Phe Glu Tyr Leu Phe Glu Gln Gly Lys His Glu Phe
145 150 155 160
Val Asn Asp Val Ala Ser Leu Trp Glu Leu Ser Thr Glu Glu Glu Ile
165 170 175
His His Phe Lys Asn Glu Ser Leu Gly Met Ala Phe Leu His Leu Cys
180 185 190
His Leu Ala Leu Arg His Gly Ile Pro Leu Glu Glu Val Ala Lys Lys
195 200 205
Thr Ser Phe Lys Asp Cys Ile Pro Arg Ser Phe Arg Arg His Ile Arg
210 215 220
Gln His Ser Ala Leu Thr Arg Leu Arg Leu Arg Asn Val Phe Arg Arg
225 230235 240
Phe Leu Arg Asp Phe Gln Pro Gly Arg Leu Ser Gln Gln Met Val Met
245 250 255
Val Lys Tyr Leu Ala Thr Leu Glu Arg Leu Ala Pro Arg Phe Gly Thr
260 265 270
Glu Arg Val Pro Val Cys His Leu Arg Leu Leu Ala Gln Ala Glu Gly
275 280 285
Glu Pro Cys Tyr Ile Arg Asp Ser Gly Val Ala Pro Thr Asp Pro Gly
290 295 300
Pro Glu Ser Ala Ala Gly Pro Pro Thr His Glu Val Leu Val Thr Gly
305 310 315 320
Thr Gly Gly Ile Gln Trp Trp Pro Val Glu Glu Glu Val Asn Lys Glu
325 330 335
Glu Gly Ser Ser Gly Ser Ser Gly Arg Asn Pro Gln Ala Ser Leu Phe
340 345 350
Gly Lys Lys Ala Lys Ala His Lys Ala Val Gly Gln Pro Ala Asp Arg
355 360 365
Pro Arg Glu Pro Leu Trp Ala Tyr Phe Cys Asp Phe Arg Asp Ile Thr
370 375 380
His Val Val Leu Lys Glu His Cys Val Ser Ile His Arg Gln Asp Asn
385 390 395 400
Lys Cys Leu Glu Leu Ser Leu Pro Ser Arg Ala Ala Ala Leu Ser Phe
405 410 415
Val Ser Leu Val Asp Gly Tyr Phe Arg Leu Thr Ala Asp Ser Ser His
420 425 430
Tyr Leu Cys His Glu Val Ala Pro Pro Arg Leu Val Met Ser Ile Arg
435 440 445
Asp Gly Ile His Gly Pro Leu Leu Glu Pro Phe Val Gln Ala Lys Leu
450 455 460
Arg Pro Glu Asp Gly Leu Tyr Leu Ile His Trp Ser Thr Ser His Pro
465 470 475 480
Tyr Arg Leu Ile Leu Thr Val Ala Gln Arg Ser Gln Ala Pro Asp Gly
485 490 495
Met Gln Ser Leu Arg Leu Arg Lys Phe Pro Ile Glu Gln Gln Asp Gly
500 505 510
Ala Phe Val Leu Glu Gly Trp Gly Arg Ser Phe Pro Ser Val Arg Glu
515 520 525
Leu Gly Ala Ala Leu Gln Gly Cys Leu Leu Arg Ala Gly Asp Asp Cys
530 535 540
Phe Ser Leu Arg Arg Cys Cys Leu Pro Gln Pro Gly Glu Thr Ser Asn
545 550 555 560
Leu Ile Ile Met Arg Gly Ala Arg Ala Ser Pro Arg Thr Leu Asn Leu
565 570 575
Ser Gln Leu Ser Phe His Arg Val Asp Gln Lys Glu Ile Thr Gln Leu
580 585 590
Ser His Leu Gly Gln Gly Thr Arg Thr Asn Val Tyr Glu Gly Arg Leu
595 600 605
Arg Val Glu Gly Ser Gly Asp Pro Glu Glu Gly Lys Met Asp Asp Glu
610 615 620
Asp Pro Leu Val Pro Gly Arg Asp Arg Gly Gln Glu Leu Arg Val Val
625 630 635 640
Leu Lys Val Leu Asp Pro Ser His His Asp Ile Ala Leu Ala Phe Tyr
645 650 655
Glu Thr Ala Ser Leu Met Ser Gln Val Ser His Thr His Leu Ala Phe
660 665 670
Val His Gly Val Cys Val Arg Gly Pro Glu Asn Ile Met Val Thr Glu
675 680 685
Tyr Val Glu His Gly Pro Leu Asp Val Trp Leu Arg Arg Glu Arg Gly
690 695 700
His Val Pro Met Ala Trp Lys Met Val Val Ala Gln Gln Leu Ala Ser
705 710 715 720
Ala Leu Ser Tyr Leu Glu Asn Lys Asn Leu Val His Gly Asn Val Cys
725 730 735
Gly Arg Asn Ile Leu Leu Ala Arg Leu Gly Leu Ala Glu Gly Thr Ser
740 745 750
Pro Phe Ile Lys Leu Ser Asp Pro Gly Val Gly Leu Gly Ala Leu Ser
755 760 765
Arg Glu Glu Arg Val Glu Arg Ile Pro Trp Leu Ala Pro Glu Cys Leu
770 775 780
Pro Gly Gly Ala Asn Ser Leu Ser Thr Ala Met Asp Lys Trp Gly Phe
785 790795 800
Gly Ala Thr Leu Leu Glu Ile Cys Phe Asp Gly Glu Ala Pro Leu Gln
805 810 815
Ser Arg Ser Pro Ser Glu Lys Glu His Phe Tyr Gln Arg Gln His Arg
820 825 830
Leu Pro Glu Pro Ser Cys Pro Gln Leu Ala Thr Leu Thr Ser Gln Cys
835 840 845
Leu Thr Tyr Glu Pro Thr Gln Arg Pro Ser Phe Arg Thr Ile Leu Arg
850 855 860
Asp Leu Thr Arg Leu Gln Pro His Asn Leu Ala Asp Val Leu Thr Val
865 870 875 880
Asn Pro Asp Ser Pro Ala Ser Asp Pro Thr Val Phe His Lys Arg Tyr
885 890 895
Leu Lys Lys Ile Arg Asp Leu Gly Glu Gly His Phe Gly Lys Val Ser
900 905 910
Leu Tyr Cys Tyr Asp Pro Thr Asn Asp Gly Thr Gly Glu Met Val Ala
915 920 925
Val Lys Ala Leu Lys Ala Asp Cys Gly Pro Gln His Arg Ser Gly Trp
930 935 940
Lys Gln Glu Ile Asp Ile Leu Arg Thr Leu Tyr His Glu His Ile Ile
945 950 955 960
Lys Tyr Lys Gly Cys Cys Glu Asp Gln Gly Glu Lys Ser Leu Gln Leu
965 970 975
Val Met Glu Tyr Val Pro Leu Gly Ser Leu Arg Asp Tyr Leu Pro Arg
980 985 990
His Ser Ile Gly Leu Ala Gln Leu Leu Leu Phe Ala Gln Gln Ile Cys
995 1000 1005
Glu Gly Met Ala Tyr Leu His Ala Gln His Tyr Ile His Arg Asp Leu
1010 1015 1020
Ala Ala Arg Asn Val Leu Leu Asp Asn Asp Arg Leu Val Lys Ile Gly
1025 1030 1035 1040
Asp Phe Gly Leu Ala Lys Ala Val Pro Glu Gly His Glu Tyr Tyr Arg
1045 1050 1055
Val Arg Glu Asp Gly Asp Ser Pro Val Phe Trp Tyr Ala Pro Glu Cys
1060 1065 1070
Leu Lys Glu Tyr Lys Phe Tyr Tyr Ala Ser Asp Val Trp Ser Phe Gly
1075 1080 1085
Val Thr Leu Tyr Glu Leu Leu Thr His Cys Asp Ser Ser Gln Ser Pro
1090 1095 1100
Pro Thr Lys Phe Leu Glu Leu Ile Gly Ile Ala Gln Gly Gln Met Thr
1105 1110 1115 1120
Val Leu Arg Leu Thr Glu Leu Leu Glu Arg Gly Glu Arg Leu Pro Arg
1125 1130 1135
Pro Asp Lys Cys Pro Cys Glu Val Tyr His Leu Met Lys Asn Cys Trp
1140 1145 1150
Glu Thr Glu Ala Ser Phe Arg Pro Thr Phe Glu Asn Leu Ile Pro Ile
1155 1160 1165
Leu Lys Thr Val His Glu Lys Tyr Gln Gly Gln Ala Pro Ser Val Phe
1170 1175 1180
Ser Val Cys
1185
<210>57
<211>906
<212>PRT
<213> human (Homo sapiens)
<400>57
Met Ala Gly Asn Val Lys Lys Ser Ser Gly Ala Gly Gly Gly Ser Gly
1 5 10 15
Ser Gly Gly Ser Gly Ser Gly Gly Leu Ile Gly Leu Met Lys Asp Ala
20 25 30
Phe Gln Pro His His His His His His His Leu Ser Pro His Pro Pro
35 40 45
Gly Thr Val Asp Lys Lys Met Val Glu Lys Cys Trp Lys Leu Met Asp
50 55 60
Lys Val Val Arg Leu Cys Gln Asn Pro Lys Leu Ala Leu Lys Asn Ser
65 70 75 80
Pro Pro Tyr Ile Leu Asp Leu Leu Pro Asp Thr Tyr Gln His Leu Arg
85 90 95
Thr Ile Leu Ser Arg Tyr Glu Gly Lys Met Glu Thr Leu Gly Glu Asn
100 105 110
Glu Tyr Phe Arg Val Phe Met Glu Asn Leu Met Lys Lys Thr Lys Gln
115 120 125
Thr Ile Ser Leu Phe Lys Glu Gly Lys Glu Arg Met Tyr Glu Glu Asn
130 135 140
Ser Gln Pro Arg Arg Asn Leu Thr Lys Leu Ser Leu Ile Phe Ser His
145 150 155 160
Met Leu Ala Glu Leu Lys Gly Ile Phe Pro Ser Gly Leu Phe Gln Gly
165 170 175
Asp Thr Phe Arg Ile Thr Lys Ala Asp Ala Ala Glu Phe Trp Arg Lys
180 185 190
Ala Phe Gly Glu Lys Thr Ile Val Pro Trp Lys Ser Phe Arg Gln Ala
195 200 205
Leu His Glu Val His Pro Ile Ser Ser Gly Leu Glu Ala Met Ala Leu
210 215 220
Lys Ser Thr Ile Asp Leu Thr Cys Asn Asp Tyr Ile Ser Val Phe Glu
225 230 235 240
Phe Asp Ile Phe Thr Arg Leu Phe Gln Pro Trp Ser Ser Leu Leu Arg
245 250 255
Asn Trp Asn Ser Leu Ala Val Thr His Pro Gly Tyr Met Ala Phe Leu
260 265 270
Thr Tyr Asp Glu Val Lys Ala Arg Leu Gln Lys Phe Ile His Lys Pro
275 280 285
Gly Ser Tyr Ile Phe Arg Leu Ser Cys Thr Arg Leu Gly Gln Trp Ala
290 295 300
Ile Gly Tyr Val Thr Ala Asp Gly Asn Ile Leu Gln Thr Ile Pro His
305 310 315 320
Asn Lys Pro Leu Phe Gln Ala Leu Ile Asp Gly Phe Arg Glu Gly Phe
325 330 335
Tyr Leu Phe Pro Asp Gly Arg Asn Gln Asn Pro Asp Leu Thr Gly Leu
340 345 350
Cys Glu Pro Thr Pro Gln Asp His Ile Lys Val Thr Gln Glu Gln Tyr
355 360 365
Glu Leu Tyr Cys Glu Met Gly Ser Thr Phe Gln Leu Cys Lys Ile Cys
370 375 380
Ala Glu Asn Asp Lys Asp Val Lys Ile Glu Pro Cys Gly His Leu Met
385 390 395 400
Cys Thr Ser Cys Leu Thr Ser Trp Gln Glu Ser Glu Gly Gln Gly Cys
405 410 415
Pro Phe Cys Arg Cys Glu Ile Lys Gly Thr Glu Pro Ile Val Val Asp
420 425 430
Pro Phe Asp Pro Arg Gly Ser Gly Ser Leu Leu Arg Gln Gly Ala Glu
435 440 445
Gly Ala Pro Ser Pro Asn Tyr Asp Asp Asp Asp Asp Glu Arg Ala Asp
450 455 460
Asp Thr Leu Phe Met Met Lys Glu Leu Ala Gly Ala Lys Val Glu Arg
465 470 475 480
Pro Pro Ser Pro Phe Ser Met Ala Pro Gln Ala Ser Leu Pro Pro Val
485 490 495
Pro Pro Arg Leu Asp Leu Leu Pro Gln Arg Val Cys Val Pro Ser Ser
500 505 510
Ala Ser Ala Leu Gly Thr Ala Ser Lys Ala Ala Ser Gly Ser Leu His
515 520 525
Lys Asp Lys Pro Leu Pro Val Pro Pro Thr Leu Arg Asp Leu Pro Pro
530 535 540
Pro Pro Pro Pro Asp Arg Pro Tyr Ser Val Gly Ala Glu Ser Arg Pro
545 550 555560
Gln Arg Arg Pro Leu Pro Cys Thr Pro Gly Asp Cys Pro Ser Arg Asp
565 570 575
Lys Leu Pro Pro Val Pro Ser Ser Arg Leu Gly Asp Ser Trp Leu Pro
580 585 590
Arg Pro Ile Pro Lys Val Pro Val Ser Ala Pro Ser Ser Ser Asp Pro
595 600 605
Trp Thr Gly Arg Glu Leu Thr Asn Arg His Ser Leu Pro Phe Ser Leu
610 615 620
Pro Ser Gln Met Glu Pro Arg Pro Asp Val Pro Arg Leu Gly Ser Thr
625 630 635 640
Phe Ser Leu Asp Thr Ser Met Ser Met Asn Ser Ser Pro Leu Val Gly
645 650 655
Pro Glu Cys Asp His Pro Lys Ile Lys Pro Ser Ser Ser Ala Asn Ala
660 665 670
Ile Tyr Ser Leu Ala Ala Arg Pro Leu Pro Val Pro Lys Leu Pro Pro
675 680 685
Gly Glu Gln Cys Glu Gly Glu Glu Asp Thr Glu Tyr Met Thr Pro Ser
690 695 700
Ser Arg Pro Leu Arg Pro Leu Asp Thr Ser Gln Ser Ser Arg Ala Cys
705 710 715 720
Asp Cys Asp Gln Gln Ile Asp Ser Cys Thr Tyr Glu Ala Met Tyr Asn
725 730 735
Ile Gln Ser Gln Ala Pro Ser Ile Thr Glu Ser Ser Thr Phe Gly Glu
740 745 750
Gly Asn Leu Ala Ala Ala His Ala Asn Thr Gly Pro Glu Glu Ser Glu
755 760 765
Asn Glu Asp Asp Gly Tyr Asp Val Pro Lys Pro Pro Val Pro Ala Val
770 775 780
Leu Ala Arg Arg Thr Leu Ser Asp Ile Ser Asn Ala Ser Ser Ser Phe
785 790 795 800
Gly Trp Leu Ser Leu Asp Gly Asp Pro Thr Thr Asn Val Thr Glu Gly
805 810 815
Ser Gln Val Pro Glu Arg Pro Pro Lys Pro Phe Pro Arg Arg Ile Asn
820 825 830
Ser Glu Arg Lys Ala Gly Ser Cys Gln Gln Gly Ser Gly Pro Ala Ala
835 840 845
Ser Ala Ala Thr Ala Ser Pro Gln Leu Ser Ser Glu Ile Glu Asn Leu
850 855 860
Met Ser Gln Gly Tyr Ser Tyr Gln Asp Ile Gln Lys Ala Leu Val Ile
865 870 875 880
Ala Gln Asn Asn Ile Glu Met Ala Lys Asn Ile Leu Arg Glu Phe Val
885 890 895
Ser Ile Ser Ser Pro Ala His Val Ala Thr
900 905
<210>58
<211>465
<212>PRT
<213> human (Homo sapiens)
<400>58
Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu
1 5 10 15
Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys
20 25 30
Arg Gln Gly Val Pro Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys
35 40 45
Glu Leu Arg Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln
50 55 60
Ser Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met
65 70 75 80
Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu
85 90 95
Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu
100 105 110
Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg
115 120 125
Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn
130 135 140
Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly
145 150 155 160
Lys Leu Arg Val Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu
165 170 175
Thr Gln Gly Pro Ser Cys Trp Asp Asp Val Leu Ile Pro Asn Arg Met
180 185 190
Ser Gly Glu Cys Gln Ser Pro His Cys Pro Gly Thr Ser Ala Glu Phe
195 200 205
Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys Glu Thr Ser Val
210 215 220
Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr
225 230 235 240
Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg
245 250 255
His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu
260 265 270
Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro
275 280 285
Cys Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His His Phe
290 295 300
Arg Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala
305 310 315 320
Glu GluCys Val Leu Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly
325 330 335
Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys
340 345 350
Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys
355 360 365
Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser
370 375 380
Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala Glu Gln
385 390 395 400
Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys
405 410 415
Pro Cys Pro Arg Cys His Val Pro Val Glu Lys Asn Gly Gly Cys Met
420 425 430
His Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn
435 440 445
Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp
450 455 460
Val
465
<210>59
<211>108
<212>PRT
<213> human (Homo sapiens)
<400>59
Met Ala Ala Ala Met Asp Val Asp Thr Pro Ser Gly Thr Asn Ser Gly
1 5 10 15
Ala Gly Lys Lys Arg Phe Glu Val Lys Lys Trp Asn Ala Val Ala Leu
20 25 30
Trp Ala Trp Asp Ile Val Val Asp Asn Cys Ala Ile Cys Arg Asn His
35 40 45
Ile Met Asp Leu Cys Ile Glu Cys Gln Ala Asn Gln Ala Ser Ala Thr
50 55 60
Ser Glu Glu Cys Thr Val Ala Trp Gly Val Cys Asn His Ala Phe His
65 70 75 80
Phe His Cys Ile Ser Arg Trp Leu Lys Thr Arg Gln Val Cys Pro Leu
85 90 95
Asp Asn Arg Glu Trp Glu Phe Gln Lys Tyr Gly His
100 105
<210>60
<211>501
<212>PRT
<213> human (Homo sapiens)
<400>60
Met Ala Ala Ala Ser Val Thr Pro Pro Gly Ser Leu Glu Leu Leu Gln
1 5 10 15
Pro Gly Phe Ser Lys Thr Leu Leu Gly Thr Lys Leu Glu Ala Lys Tyr
20 25 30
Leu Cys Ser Ala Cys Arg Asn Val Leu Arg Arg Pro Phe Gln Ala Gln
35 40 45
Cys Gly His Arg Tyr Cys Ser Phe Cys Leu Ala Ser Ile Leu Ser Ser
50 55 60
Gly Pro Gln Asn Cys Ala Ala Cys Val His Glu Gly Ile Tyr Glu Glu
65 70 75 80
Gly Ile Ser Ile Leu Glu Ser Ser Ser Ala Phe Pro Asp Asn Ala Ala
85 90 95
Arg Arg Glu Val Glu Ser Leu Pro Ala Val Cys Pro Ser Asp Gly Cys
100 105110
Thr Trp Lys Gly Thr Leu Lys Glu Tyr Glu Ser Cys His Glu Gly Arg
115 120 125
Cys Pro Leu Met Leu Thr Glu Cys Pro Ala Cys Lys Gly Leu Val Arg
130 135 140
Leu Gly Glu Lys Glu Arg His Leu Glu His Glu Cys Pro Glu Arg Ser
145 150 155 160
Leu Ser Cys Arg His Cys Arg Ala Pro Cys Cys Gly Ala Asp Val Lys
165 170 175
Ala His His Glu Val Cys Pro Lys Phe Pro Leu Thr Cys Asp Gly Cys
180 185 190
Gly Lys Lys Lys Ile Pro Arg Glu Lys Phe Gln Asp His Val Lys Thr
195 200 205
Cys Gly Lys Cys Arg Val Pro Cys Arg Phe His Ala Ile Gly Cys Leu
210 215 220
Glu Thr Val Glu Gly Glu Lys Gln Gln Glu His Glu Val Gln Trp Leu
225 230 235 240
Arg Glu His Leu Ala Met Leu Leu Ser Ser Val Leu Glu Ala Lys Pro
245 250 255
Leu Leu Gly Asp Gln Ser His Ala Gly Ser Glu Leu Leu Gln Arg Cys
260 265 270
Glu Ser Leu Glu Lys Lys Thr Ala Thr Phe Glu Asn Ile Val Cys Val
275 280 285
Leu Asn Arg Glu Val Glu Arg Val Ala Met Thr Ala Glu Ala Cys Ser
290 295 300
Arg Gln His Arg Leu Asp Gln Asp Lys Ile Glu Ala Leu Ser Ser Lys
305 310 315 320
Val Gln Gln Leu Glu Arg Ser Ile Gly Leu Lys Asp Leu Ala Met Ala
325 330 335
Asp Leu Glu Gln Lys Val Leu Glu Met Glu Ala Ser Thr Tyr Asp Gly
340 345 350
Val Phe Ile Trp Lys Ile Ser Asp Phe Ala Arg Lys Arg Gln Glu Ala
355 360 365
Val Ala Gly Arg Ile Pro Ala Ile Phe Ser Pro Ala Phe Tyr Thr Ser
370 375 380
Arg Tyr Gly Tyr Lys Met Cys Leu Arg Ile Tyr Leu Asn Gly Asp Gly
385 390 395 400
Thr Gly Arg Gly Thr His Leu Ser Leu Phe Phe Val Val Met Lys Gly
405 410 415
Pro Asn Asp Ala Leu Leu Arg Trp Pro Phe Asn Gln Lys Val Thr Leu
420 425 430
Met Leu Leu Asp Gln Asn Asn Arg Glu His Val Ile Asp Ala Phe Arg
435 440 445
Pro Asp Val Thr Ser Ser Ser Phe Gln Arg Pro Val Asn Asp Met Asn
450 455 460
Ile Ala Ser Gly Cys Pro Leu Phe Cys Pro Val Ser Lys Met Glu Ala
465 470 475 480
Lys Asn Ser Tyr Val Arg Asp Asp Ala Ile Phe Ile Lys Ala Ile Val
485 490 495
Asp Leu Thr Gly Leu
500
<210>61
<211>491
<212>PRT
<213> human (Homo sapiens)
<400>61
Met Cys Asn Thr Asn Met Ser Val Pro Thr Asp Gly Ala Val Thr Thr
1 5 10 15
Ser Gln Ile Pro Ala Ser Glu Gln Glu Thr Leu Val Arg Pro Lys Pro
20 25 30
Leu Leu Leu Lys Leu Leu Lys Ser Val Gly Ala Gln Lys Asp Thr Tyr
35 40 45
Thr Met Lys Glu Val Leu Phe Tyr Leu Gly Gln Tyr Ile Met Thr Lys
50 55 60
Arg Leu Tyr Asp Glu Lys Gln Gln His Ile Val Tyr Cys Ser Asn Asp
65 70 75 80
Leu Leu Gly Asp Leu Phe Gly Val Pro Ser Phe Ser Val Lys Glu His
85 90 95
Arg Lys Ile Tyr Thr Met Ile Tyr Arg Asn Leu Val Val Val Asn Gln
100 105 110
Gln Glu Ser Ser Asp Ser Gly Thr Ser Val Ser Glu Asn Arg Cys His
115 120 125
Leu Glu Gly Gly Ser Asp Gln Lys Asp Leu Val Gln Glu Leu Gln Glu
130 135 140
Glu Lys Pro Ser Ser Ser His Leu Val Ser Arg Pro Ser Thr Ser Ser
145 150 155 160
Arg Arg Arg Ala Ile Ser Glu Thr Glu Glu Asn Ser Asp Glu Leu Ser
165 170 175
Gly Glu Arg Gln Arg Lys Arg His Lys Ser Asp Ser Ile Ser Leu Ser
180 185 190
Phe Asp Glu Ser Leu Ala Leu Cys Val Ile Arg Glu Ile Cys Cys Glu
195 200 205
Arg Ser Ser Ser Ser Glu Ser Thr Gly Thr Pro Ser Asn Pro Asp Leu
210 215 220
Asp Ala Gly Val Ser Glu His Ser Gly Asp Trp Leu Asp Gln Asp Ser
225 230 235 240
Val Ser Asp Gln Phe Ser Val Glu Phe Glu Val Glu Ser Leu Asp Ser
245 250 255
Glu Asp Tyr Ser Leu Ser Glu Glu Gly Gln Glu Leu Ser Asp Glu Asp
260 265 270
Asp Glu Val Tyr Gln Val Thr Val Tyr Gln AlaGly Glu Ser Asp Thr
275 280 285
Asp Ser Phe Glu Glu Asp Pro Glu Ile Ser Leu Ala Asp Tyr Trp Lys
290 295 300
Cys Thr Ser Cys Asn Glu Met Asn Pro Pro Leu Pro Ser His Cys Asn
305 310 315 320
Arg Cys Trp Ala Leu Arg Glu Asn Trp Leu Pro Glu Asp Lys Gly Lys
325 330 335
Asp Lys Gly Glu Ile Ser Glu Lys Ala Lys Leu Glu Asn Ser Thr Gln
340 345 350
Ala Glu Glu Gly Phe Asp Val Pro Asp Cys Lys Lys Thr Ile Val Asn
355 360 365
Asp Ser Arg Glu Ser Cys Val Glu Glu Asn Asp Asp Lys Ile Thr Gln
370 375 380
Ala Ser Gln Ser Gln Glu Ser Glu Asp Tyr Ser Gln Pro Ser Thr Ser
385 390 395 400
Ser Ser Ile Ile Tyr Ser Ser Gln Glu Asp Val Lys Glu Phe Glu Arg
405 410 415
Glu Glu Thr Gln Asp Lys Glu Glu Ser Val Glu Ser Ser Leu Pro Leu
420 425 430
Asn Ala Ile Glu Pro Cys Val Ile Cys Gln Gly Arg Pro Lys Asn Gly
435 440 445
Cys Ile Val His Gly Lys Thr Gly His Leu Met Ala Cys Phe Thr Cys
450 455 460
Ala Lys Lys Leu Lys Lys Arg Asn Lys Pro Cys Pro Val Cys Arg Gln
465 470 475 480
Pro Ile Gln Met Ile Val Leu Thr Tyr Phe Pro
485 490
<210>62
<211>240
<212>PRT
<213> Renilla
<400>62
Met Thr Ser Lys Val Tyr Asp Pro Glu Gln Arg Lys Arg Met Ile Thr
1 5 10 15
Gly Pro Gln Trp Trp Ala Arg Cys Lys Gln Met Asn Val Leu Asp Ser
20 25 30
Phe Ile Asn Tyr Tyr Asp Ser Glu Lys His Ala Glu Asn Ala Val Ile
35 40 45
Phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg His Val Val
50 55 60
Pro His Ile Glu Pro Val Ala Arg Cys Ile Ile Pro Asp Leu Ile Gly
65 70 75 80
Met Gly Lys Ser Gly Lys Ser Gly Asn Gly Ser Tyr Arg Leu Leu Asp
85 90 95
His Tyr Lys Tyr Leu Thr Ala Trp Phe Glu Leu Leu Asn Leu Pro Lys
100 105 110
Lys Ile Ile Phe Val Gly His Asp Trp Gly Ala Cys Leu Ala Phe His
115 120 125
Tyr Ser Tyr Glu His Gln Asp Lys Ile Lys Ala Ile Val His Ala Glu
130 135 140
Ser Val Val Asp Val Ile Glu Ser Trp Asp Glu Trp Pro Asp Ile Glu
145 150 155 160
Glu Asp Ile Ala Leu Ile Lys Ser Glu Glu Gly Glu Lys Met Val Leu
165 170 175
Glu Asn AsnPhe Phe Val Glu Thr Met Leu Pro Ser Lys Ile Met Arg
180 185 190
Lys Leu Glu Pro Glu Glu Phe Ala Ala Tyr Leu Glu Pro Phe Lys Glu
195 200 205
Lys Gly Glu Val Arg Arg Pro Thr Leu Ser Trp Pro Arg Glu Ile Pro
210 215 220
Leu Val Lys Gly Gly Lys Pro Asp Val Val Gln Ile Val Arg Asn Tyr
225 230 235 240
<210>63
<211>320
<212>PRT
<213> human (Homo sapiens)
<400>63
Met Glu Leu Leu Ser Pro Pro Leu Arg Asp Val Asp Leu Thr Ala Pro
1 5 10 15
Asp Gly Ser Leu Cys Ser Phe Ala Thr Thr Asp Asp Phe Tyr Asp Asp
20 25 30
Pro Cys Phe Asp Ser Pro Asp Leu Arg Phe Phe Glu Asp Leu Asp Pro
35 40 45
Arg Leu Met His Val Gly Ala Leu Leu Lys Pro GluGlu His Ser His
50 55 60
Phe Pro Ala Ala Val His Pro Ala Pro Gly Ala Arg Glu Asp Glu His
65 70 75 80
Val Arg Ala Pro Ser Gly His His Gln Ala Gly Arg Cys Leu Leu Trp
85 90 95
Ala Cys Lys Ala Cys Lys Arg Lys Thr Thr Asn Ala Asp Arg Arg Lys
100 105 110
Ala Ala Thr Met Arg Glu Arg Arg Arg Leu Ser Lys Val Asn Glu Ala
115 120 125
Phe Glu Thr Leu Lys Arg Cys Thr Ser Ser Asn Pro Asn Gln Arg Leu
130 135 140
Pro Lys Val Glu Ile Leu Arg Asn Ala Ile Arg Tyr Ile Glu Gly Leu
145 150 155 160
Gln Ala Leu Leu Arg Asp Gln Asp Ala Ala Pro Pro Gly Ala Ala Ala
165 170 175
Ala Phe Tyr Ala Pro Gly Pro Leu Pro Pro Gly Arg Gly Gly Glu His
180 185 190
Tyr Ser Gly Asp Ser Asp Ala Ser Ser Pro Arg Ser Asn Cys Ser Asp
195 200 205
Gly Met Met Asp Tyr Ser Gly Pro Pro Ser Gly Ala Arg Arg Arg Asn
210 215 220
Cys Tyr Glu Gly Ala Tyr Tyr Asn Glu Ala Pro Ser Glu Pro Arg Pro
225 230 235 240
Gly Lys Ser Ala Ala Val Ser Ser Leu Asp Cys Leu Ser Ser Ile Val
245 250 255
Glu Arg Ile Ser Thr Glu Ser Pro Ala Ala Pro Ala Leu Leu Leu Ala
260 265 270
Asp Val Pro Ser Glu Ser Pro Pro Arg Arg Gln Glu Ala Ala Ala Pro
275 280 285
Ser Glu Gly Glu Ser Ser Gly Asp Pro Thr Gln Ser Pro Asp Ala Ala
290 295 300
Pro Gln Cys Pro Ala Gly Ala Asn Pro Asn Pro Ile Tyr Gln Val Leu
305 310 315 320
<210>64
<211>607
<212>PRT
<213> human (Homo sapiens)
<400>64
Met Ala Glu Lys Arg Arg Gly Ser Pro Cys Ser Met Leu Ser Leu Lys
1 5 10 15
Ala His Ala Phe Ser Val Glu Ala Leu Ile Gly Ala Glu Lys Gln Gln
20 25 30
Gln Leu Gln Lys Lys Arg Arg Lys Leu Gly Ala Glu Glu Ala Ala Gly
35 40 45
Ala Val Asp Asp Gly Gly Cys Ser Arg Gly Gly Gly Ala Gly Glu Lys
50 55 60
Gly Ser Ser Glu Gly Asp Glu Gly Ala Ala Leu Pro Pro Pro Ala Gly
65 70 75 80
Ala Thr Ser Gly Pro Ala Arg Ser Gly Ala Asp Leu Glu Arg Gly Ala
85 90 95
Ala Gly Gly Cys Glu Asp Gly Phe Gln Gln Gly Ala Ser Pro Leu Ala
100 105 110
Ser Pro Gly Gly Ser Pro Lys Gly Ser Pro Ala Arg Ser Leu Ala Arg
115 120 125
Pro Gly Thr Pro Leu Pro Ser Pro Gln Ala Pro Arg Val Asp Leu Gln
130 135 140
Gly Ala Glu Leu Trp Lys Arg Phe His Glu Ile Gly Thr Glu Met Ile
145 150 155 160
Ile Thr Lys Ala Gly Arg Arg Met Phe Pro Ala Met Arg Val Lys Ile
165 170 175
Ser Gly Leu Asp Pro His Gln Gln Tyr Tyr Ile Ala Met Asp Ile Val
180 185 190
Pro Val Asp Asn Lys Arg Tyr Arg Tyr Val Tyr His Ser Ser Lys Trp
195 200 205
Met Val Ala Gly Asn Ala Asp Ser Pro Val Pro Pro Arg Val Tyr Ile
210 215 220
His Pro Asp Ser Pro Ala Ser Gly Glu Thr Trp Met Arg Gln Val Ile
225 230 235 240
Ser Phe Asp Lys Leu Lys Leu Thr Asn Asn Glu Leu Asp Asp Gln Gly
245 250 255
His Ile Ile Leu His Ser Met His Lys Tyr Gln Pro Arg Val His Val
260 265 270
Ile Arg Lys Asp Cys Gly Asp Asp Leu Ser Pro Ile Lys Pro Val Pro
275 280 285
Ser Gly Glu Gly Val Lys Ala Phe Ser Phe Pro Glu Thr Val Phe Thr
290 295 300
Thr Val Thr Ala Tyr Gln Asn Gln Gln Ile Thr Arg Leu Lys Ile Asp
305 310 315 320
Arg Asn Pro Phe Ala Lys Gly Phe Arg Asp Ser Gly Arg Asn Arg Met
325 330 335
Gly Leu Glu Ala Leu Val Glu Ser Tyr Ala Phe Trp Arg Pro Ser Leu
340 345 350
Arg Thr Leu Thr Phe Glu Asp Ile Pro Gly Ile Pro Lys Gln Gly Asn
355 360 365
Ala Ser Ser Ser Thr Leu Leu Gln Gly Thr Gly Asn Gly Val Pro Ala
370 375 380
Thr His Pro His Leu Leu Ser Gly Ser Ser Cys Ser Ser Pro Ala Phe
385 390 395 400
His Leu Gly Pro Asn Thr Ser Gln Leu Cys Ser Leu Ala Pro Ala Asp
405 410 415
Tyr Ser Ala Cys Ala Arg Ser Gly Leu Thr Leu Asn Arg Tyr Ser Thr
420 425 430
Ser Leu Ala Glu Thr Tyr Asn Arg Leu Thr Asn Gln Ala Gly Glu Thr
435 440 445
Phe Ala Pro Pro Arg Thr Pro Ser Tyr Val Gly Val Ser Ser Ser Thr
450 455 460
Ser Val Asn Met Ser Met Gly Gly Thr Asp Gly Asp Thr Phe Ser Cys
465 470 475 480
Pro Gln Thr Ser Leu Ser Met Gln Ile Ser Gly Met Ser Pro Gln Leu
485 490 495
Gln Tyr Ile Met Pro Ser Pro Ser Ser Asn Ala Phe Ala Thr Asn Gln
500 505 510
Thr His Gln Gly Ser Tyr Asn Thr Phe Arg Leu His Ser Pro Cys Ala
515 520 525
Leu Tyr Gly Tyr Asn Phe Ser Thr Ser Pro Lys Leu Ala Ala Ser Pro
530 535 540
Glu Lys Ile Val Ser Ser Gln GlySer Phe Leu Gly Ser Ser Pro Ser
545 550 555 560
Gly Thr Met Thr Asp Arg Gln Met Leu Pro Pro Val Glu Gly Val His
565 570 575
Leu Leu Ser Ser Gly Gly Gln Gln Ser Phe Phe Asp Ser Arg Thr Leu
580 585 590
Gly Ser Leu Thr Leu Ser Ser Ser Gln Val Ser Ala His Met Val
595 600 605
<210>65
<211>393
<212>PRT
<213> human (Homo sapiens)
<400>65
Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln
1 5 10 15
Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu
20 25 30
Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp
35 40 45
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu AlaPro
50 55 60
Arg Met Pro Glu Ala Ala Pro Pro Val Ala Pro Ala Pro Ala Ala Pro
65 70 75 80
Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser
85 90 95
Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly
100 105 110
Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro
115 120 125
Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln
130 135 140
Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met
145 150 155 160
Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys
165 170 175
Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln
180 185 190
His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp
195 200 205
Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu
210 215 220
Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
225 230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr
245 250 255
Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val
260 265 270
Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn
275 280 285
Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr
290 295 300
Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys
305 310 315 320
Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu
325 330 335
Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp
340 345 350
Ala Gln Ala Gly Lys Glu Pro Gly Gly Ser Arg Ala His Ser Ser His
355 360 365
Leu Lys Ser Lys Lys Gly Gln Ser Thr Ser Arg His Lys Lys Leu Met
370 375 380
Phe Lys Thr Glu Gly Pro Asp Ser Asp
385 390
<210>66
<211>215
<212>PRT
<213> human (Homo sapiens)
<400>66
Met Gly Lys Gly Asp Pro Lys Lys Pro Arg Gly Lys Met Ser Ser Tyr
1 5 10 15
Ala Phe Phe Val Gln Thr Cys Arg Glu Glu His Lys Lys Lys His Pro
20 25 30
Asp Ala Ser Val Asn Phe Ser Glu Phe Ser Lys Lys Cys Ser Glu Arg
35 40 45
Trp Lys Thr Met Ser Ala Lys Glu Lys Gly Lys Phe Glu Asp Met Ala
50 55 60
Lys Ala Asp Lys Ala Arg Tyr Glu Arg Glu Met Lys Thr Tyr Ile Pro
65 70 75 80
Pro Lys Gly Glu Thr Lys Lys Lys Phe Lys Asp Pro Asn Ala Pro Lys
85 90 95
Arg Pro Pro Ser Ala Phe Phe Leu Phe Cys Ser Glu Tyr Arg Pro Lys
100 105 110
Ile Lys Gly Glu His Pro Gly Leu Ser Ile Gly Asp Val Ala Lys Lys
115 120 125
Leu Gly Glu Met Trp Asn Asn Thr Ala Ala Asp Asp Lys Gln Pro Tyr
130 135 140
Glu Lys Lys Ala Ala Lys Leu Lys Glu Lys Tyr Glu Lys Asp Ile Ala
145 150 155 160
Ala Tyr Arg Ala Lys Gly Lys Pro Asp Ala Ala Lys Lys Gly Val Val
165 170 175
Lys Ala Glu Lys Ser Lys Lys Lys Lys Glu Glu Glu Glu Asp Glu Glu
180 185 190
Asp Glu Glu Asp Glu Glu Glu Glu Glu Asp Glu Glu Asp Glu Asp Glu
195 200 205
Glu Glu Asp Asp Asp Asp Glu
210 215
<210>67
<211>356
<212>PRT
<213> human (Homo sapiens)
<400>67
Met Thr Lys Ser Tyr Ser Glu Ser Gly Leu Met Gly Glu Pro Gln Pro
1 5 10 15
Gln Gly Pro Pro Ser Trp Thr Asp Glu Cys Leu Ser Ser Gln Asp Glu
20 25 30
Glu His Glu Ala Asp Lys Lys Glu Asp Asp Leu Glu Thr Met Asn Ala
35 40 45
Glu Glu Asp Ser Leu Arg Asn Gly Gly Glu Glu Glu Asp Glu Asp Glu
50 55 60
Asp Leu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Asp Asp Gln Lys
65 70 75 80
Pro Lys Arg Arg Gly Pro Lys Lys Lys Lys Met Thr Lys Ala Arg Leu
85 90 95
Glu Arg Phe Lys Leu Arg Arg Met Lys Ala Asn Ala Arg Glu Arg Asn
100 105 110
Arg Met His Gly Leu Asn Ala Ala Leu Asp Asn Leu Arg Lys Val Val
115 120 125
Pro Cys Tyr Ser Lys Thr Gln Lys Leu Ser Lys Ile Glu Thr Leu Arg
130 135 140
Leu Ala Lys Asn Tyr Ile Trp Ala Leu Ser Glu Ile Leu Arg Ser Gly
145 150 155 160
Lys Ser Pro Asp Leu Val Ser Phe Val Gln Thr Leu Cys Lys Gly Leu
165 170 175
Ser Gln Pro Thr Thr Asn Leu Val Ala Gly Cys Leu Gln Leu Asn Pro
180 185 190
Arg Thr Phe Leu Pro Glu Gln Asn Gln Asp Met Pro Pro His Leu Pro
195 200 205
Thr Ala Ser Ala Ser Phe Pro Val His Pro Tyr Ser Tyr Gln Ser Pro
210 215 220
Gly Leu Pro Ser Pro Pro Tyr Gly Thr Met Asp Ser Ser His Val Phe
225 230 235 240
His Val Lys Pro Pro Pro His Ala Tyr Ser Ala Ala Leu Glu Pro Phe
245 250 255
Phe Glu Ser Pro Leu Thr Asp Cys Thr Ser Pro Ser Phe Asp Gly Pro
260 265 270
Leu Ser Pro Pro Leu Ser Ile Asn Gly Asn Phe Ser Phe Lys His Glu
275 280 285
Pro Ser Ala Glu Phe Glu Lys Asn Tyr Ala Phe Thr Met His Tyr Pro
290 295 300
Ala Ala Thr Leu Ala Gly Ala Gln Ser His Gly Ser Ile Phe Ser Gly
305 310 315 320
Thr Ala Ala Pro Arg Cys Glu Ile Pro Ile Asp Asn Ile Met Ser Phe
325 330 335
Asp Ser His Ser His His Glu Arg Val Met Ser Ala Gln Leu Asn Ala
340 345 350
Ile Phe His Asp
355
<210>68
<211>498
<212>PRT
<213> human (Homo sapiens)
<400>68
Met Asn Gln Ser Ile Pro Val Ala Pro Thr Pro Pro Arg Arg Val Arg
1 5 10 15
Leu Lys Pro Trp Leu Val Ala Gln Val Asn Ser Cys Gln Tyr Pro Gly
20 25 30
Leu Gln Trp Val Asn Gly Glu Lys Lys Leu Phe Cys Ile Pro Trp Arg
35 40 45
His Ala Thr Arg His Gly Pro Ser Gln Asp Gly Asp Asn Thr Ile Phe
50 55 60
Lys Ala Trp Ala Lys Glu Thr Gly Lys Tyr Thr Glu Gly Val Asp Glu
65 70 75 80
Ala Asp Pro Ala Lys Trp Lys Ala Asn Leu Arg Cys Ala Leu Asn Lys
85 90 95
Ser Arg Asp Phe Arg Leu Ile Tyr Asp Gly Pro Arg Asp Met Pro Pro
100 105 110
Gln Pro Tyr Lys Ile Tyr Glu Val Cys Ser Asn Gly Pro Ala Pro Thr
115 120 125
Asp Ser Gln Pro Pro Glu Asp Tyr Ser Phe Gly Ala Gly Glu Glu Glu
130 135 140
Glu Glu Glu Glu Glu Leu Gln Arg Met Leu Pro Ser Leu Ser Leu Thr
145 150 155 160
Glu Asp Val Lys Trp Pro Pro Thr Leu Gln Pro Pro Thr Leu Arg Pro
165 170 175
Pro Thr Leu Gln Pro Pro Thr Leu Gln Pro Pro Val Val Leu Gly Pro
180 185 190
Pro Ala Pro Asp Pro Ser Pro Leu Ala Pro Pro Pro Gly Asn Pro Ala
195 200 205
Gly Phe Arg Glu Leu Leu Ser Glu Val Leu Glu Pro Gly Pro Leu Pro
210 215 220
Ala Ser Leu Pro Pro Ala Gly Glu Gln Leu Leu Pro Asp Leu Leu Ile
225 230 235 240
Ser Pro His Met Leu Pro Leu Thr Asp Leu Glu Ile Lys Phe Gln Tyr
245 250 255
Arg Gly Arg Pro Pro Arg AlaLeu Thr Ile Ser Asn Pro His Gly Cys
260 265 270
Arg Leu Phe Tyr Ser Gln Leu Glu Ala Thr Gln Glu Gln Val Glu Leu
275 280 285
Phe Gly Pro Ile Ser Leu Glu Gln Val Arg Phe Pro Ser Pro Glu Asp
290 295 300
Ile Pro Ser Asp Lys Gln Arg Phe Tyr Thr Asn Gln Leu Leu Asp Val
305 310 315 320
Leu Asp Arg Gly Leu Ile Leu Gln Leu Gln Gly Gln Asp Leu Tyr Ala
325 330 335
Ile Arg Leu Cys Gln Cys Lys Val Phe Trp Ser Gly Pro Cys Ala Ser
340 345 350
Ala His Asp Ser Cys Pro Asn Pro Ile Gln Arg Glu Val Lys Thr Lys
355 360 365
Leu Phe Ser Leu Glu His Phe Leu Asn Glu Leu Ile Leu Phe Gln Lys
370 375 380
Gly Gln Thr Asn Thr Pro Pro Pro Phe Glu Ile Phe Phe Cys Phe Gly
385 390 395 400
Glu Glu Trp Pro Asp Arg Lys Pro Arg Glu Lys Lys Leu Ile Thr Val
405 410 415
Gln Val Val Pro Val Ala Ala Arg Leu Leu Leu Glu Met Phe Ser Gly
420 425 430
Glu Leu Ser Trp Ser Ala Asp Ser Ile Arg Leu Gln Ile Ser Asn Pro
435 440 445
Asp Leu Lys Asp Arg Met Val Glu Gln Phe Lys Glu Leu His His Ile
450 455 460
Trp Gln Ser Gln Gln Arg Leu Gln Pro Val Ala Gln Ala Pro Pro Gly
465 470 475 480
Ala Gly Leu Gly Val Gly Gln Gly Pro Trp Pro Met His Pro Ala Gly
485 490 495
Met Gln
<210>69
<211>427
<212>PRT
<213> human (Homo sapiens)
<400>69
Met Gly Thr Pro Lys Pro Arg Ile Leu Pro Trp Leu Val Ser Gln Leu
1 5 1015
Asp Leu Gly Gln Leu Glu Gly Val Ala Trp Val Asn Lys Ser Arg Thr
20 25 30
Arg Phe Arg Ile Pro Trp Lys His Gly Leu Arg Gln Asp Ala Gln Gln
35 40 45
Glu Asp Phe Gly Ile Phe Gln Ala Trp Ala Glu Ala Thr Gly Ala Tyr
50 55 60
Val Pro Gly Arg Asp Lys Pro Asp Leu Pro Thr Trp Lys Arg Asn Phe
65 70 75 80
Arg Ser Ala Leu Asn Arg Lys Glu Gly Leu Arg Leu Ala Glu Asp Arg
85 90 95
Ser Lys Asp Pro His Asp Pro His Lys Ile Tyr Glu Phe Val Asn Ser
100 105 110
Gly Val Gly Asp Phe Ser Gln Pro Asp Thr Ser Pro Asp Thr Asn Gly
115 120 125
Gly Gly Ser Thr Ser Asp Thr Gln Glu Asp Ile Leu Asp Glu Leu Leu
130 135 140
Gly Asn Met Val Leu Ala Pro Leu Pro Asp Pro Gly Pro Pro Ser Leu
145 150 155 160
Ala Val Ala Pro Glu Pro Cys Pro Gln Pro Leu Arg Ser Pro Ser Leu
165 170 175
Asp Asn Pro Thr Pro Phe Pro Asn Leu Gly Pro Ser Glu Asn Pro Leu
180 185 190
Lys Arg Leu Leu Val Pro Gly Glu Glu Trp Glu Phe Glu Val Thr Ala
195 200 205
Phe Tyr Arg Gly Arg Gln Val Phe Gln Gln Thr Ile Ser Cys Pro Glu
210 215 220
Gly Leu Arg Leu Val Gly Ser Glu Val Gly Asp Arg Thr Leu Pro Gly
225 230 235 240
Trp Pro Val Thr Leu Pro Asp Pro Gly Met Ser Leu Thr Asp Arg Gly
245 250 255
Val Met Ser Tyr Val Arg His Val Leu Ser Cys Leu Gly Gly Gly Leu
260 265 270
Ala Leu Trp Arg Ala Gly Gln Trp Leu Trp Ala Gln Arg Leu Gly His
275 280 285
Cys His Thr Tyr Trp Ala Val Ser Glu Glu Leu Leu Pro Asn Ser Gly
290 295 300
His Gly Pro Asp Gly Glu Val Pro Lys Asp Lys Glu Gly Gly Val Phe
305 310 315 320
Asp Leu Gly Pro Phe Ile Val Asp Leu Ile Thr Phe Thr Glu Gly Ser
325 330 335
Gly Arg Ser Pro Arg Tyr Ala Leu Trp Phe Cys Val Gly Glu Ser Trp
340 345 350
Pro Gln Asp Gln Pro Trp Thr Lys Arg Leu Val Met Val Lys Val Val
355 360 365
Pro Thr Cys Leu Arg Ala Leu Val Glu Met Ala Arg Val Gly Gly Ala
370 375 380
Ser Ser Leu Glu Asn Thr Val Asp Leu His Ile Ser Asn Ser His Pro
385 390 395 400
Leu Ser Leu Thr Ser Asp Gln Tyr Lys Ala Tyr Leu Gln Asp Leu Val
405 410 415
Glu Gly Met Asp Phe Gln Gly Pro Gly Glu Ser
420 425
<210>70
<211>750
<212>PRT
<213> human (Homo sapiens)
<400>70
Met Ser Gln Trp Tyr Glu Leu Gln Gln Leu Asp Ser Lys Phe Leu Glu
1 5 10 15
Gln Val His Gln Leu Tyr Asp Asp Ser Phe Pro Met Glu Ile Arg Gln
20 25 30
Tyr Leu Ala Gln Trp Leu Glu Lys Gln Asp Trp Glu His Ala Ala Asn
35 40 45
Asp Val Ser Phe Ala Thr Ile Arg Phe His Asp Leu Leu Ser Gln Leu
50 55 60
Asp Asp Gln Tyr Ser Arg Phe Ser Leu Glu Asn Asn Phe Leu Leu Gln
65 70 75 80
His Asn Ile Arg Lys Ser Lys Arg Asn Leu Gln Asp Asn Phe Gln Glu
85 90 95
Asp Pro Ile Gln Met Ser Met Ile Ile Tyr Ser Cys Leu Lys Glu Glu
100 105 110
Arg Lys Ile Leu Glu Asn Ala Gln Arg Phe Asn Gln Ala Gln Ser Gly
115 120 125
Asn Ile Gln Ser Thr Val Met Leu Asp Lys Gln Lys Glu Leu Asp Ser
130 135 140
Lys Val Arg Asn Val Lys Asp Lys Val Met Cys Ile Glu His Glu Ile
145 150 155 160
Lys Ser Leu Glu Asp Leu Gln Asp Glu Tyr Asp Phe Lys Cys Lys Thr
165 170 175
Leu Gln Asn Arg Glu His Glu Thr Asn Gly Val Ala Lys Ser Asp Gln
180 185 190
Lys Gln Glu Gln Leu Leu Leu Lys Lys Met Tyr Leu Met Leu Asp Asn
195 200 205
Lys Arg Lys Glu Val Val His Lys Ile Ile Glu Leu Leu Asn Val Thr
210 215 220
Glu Leu Thr Gln Asn Ala Leu Ile Asn Asp Glu Leu Val Glu Trp Lys
225 230 235 240
Arg Arg Gln Gln Ser Ala Cys Ile Gly Gly Pro Pro Asn Ala Cys Leu
245 250 255
Asp Gln Leu Gln Asn Trp Phe Thr Ile Val Ala Glu Ser Leu Gln Gln
260 265 270
Val Arg Gln Gln Leu Lys Lys Leu Glu Glu Leu Glu Gln Lys Tyr Thr
275 280 285
Tyr Glu His Asp Pro Ile Thr Lys Asn Lys Gln Val Leu Trp Asp Arg
290 295 300
Thr Phe Ser Leu Phe Gln Gln Leu Ile Gln Ser Ser Phe Val Val Glu
305 310 315 320
Arg Gln Pro Cys Met Pro Thr His Pro Gln Arg Pro Leu Val Leu Lys
325 330 335
Thr Gly Val Gln Phe Thr Val Lys Leu Arg Leu Leu Val Lys Leu Gln
340 345 350
Glu Leu Asn Tyr Asn Leu Lys Val Lys Val Leu Phe Asp Lys Asp Val
355 360 365
Asn Glu Arg Asn Thr Val Lys Gly Phe Arg Lys Phe Asn Ile Leu Gly
370 375 380
Thr His Thr Lys Val Met Asn Met Glu Glu Ser Thr Asn Gly Ser Leu
385 390 395 400
Ala Ala Glu Phe Arg His Leu Gln Leu Lys Glu Gln Lys Asn Ala Gly
405 410 415
Thr Arg Thr Asn Glu Gly Pro Leu Ile Val Thr Glu Glu Leu His Ser
420 425 430
Leu Ser Phe Glu Thr Gln Leu Cys Gln Pro Gly Leu Val Ile Asp Leu
435 440 445
Glu Thr Thr Ser Leu Pro Val Val Val Ile Ser Asn Val Ser Gln Leu
450 455 460
Pro Ser Gly Trp Ala Ser Ile Leu Trp Tyr Asn Met Leu Val Ala Glu
465 470 475 480
Pro Arg Asn Leu Ser Phe Phe Leu Thr Pro Pro Cys Ala Arg Trp Ala
485 490 495
Gln Leu Ser Glu Val Leu Ser Trp Gln Phe Ser Ser Val Thr Lys Arg
500 505 510
Gly Leu Asn Val Asp Gln Leu Asn Met Leu Gly Glu Lys Leu Leu Gly
515 520 525
Pro Asn Ala Ser Pro Asp Gly Leu Ile Pro Trp Thr Arg Phe Cys Lys
530 535 540
Glu Asn Ile Asn Asp Lys Asn Phe Pro Phe Trp Leu Trp Ile Glu Ser
545 550 555 560
Ile Leu Glu Leu Ile Lys Lys His Leu Leu Pro Leu Trp Asn Asp Gly
565 570 575
Cys Ile Met Gly Phe Ile Ser Lys Glu Arg Glu Arg Ala Leu Leu Lys
580 585 590
Asp Gln Gln Pro Gly Thr Phe Leu Leu Arg Phe Ser Glu Ser Ser Arg
595 600 605
Glu Gly Ala Ile Thr Phe Thr Trp Val Glu Arg Ser Gln Asn Gly Gly
610 615 620
Glu Pro Asp Phe His Ala Val Glu Pro Tyr Thr Lys Lys Glu Leu Ser
625 630 635 640
Ala Val Thr Phe Pro Asp Ile Ile Arg Asn Tyr Lys Val Met Ala Ala
645 650 655
Glu Asn Ile Pro Glu Asn Pro Leu Lys Tyr Leu Tyr Pro Asn Ile Asp
660 665 670
Lys Asp His Ala Phe Gly Lys Tyr Tyr Ser Arg Pro Lys Glu Ala Pro
675 680 685
Glu Pro Met Glu Leu Asp Gly Pro Lys Gly Thr Gly Tyr Ile Lys Thr
690 695 700
Glu Leu Ile Ser Val Ser Glu Val His Pro Ser Arg Leu Gln Thr Thr
705 710 715 720
Asp Asn Leu Leu Pro Met Ser Pro Glu Glu Phe Asp Glu Val Ser Arg
725 730 735
Ile Val Gly Ser Val Glu Phe Asp Ser Met Met Asn Thr Val
740 745 750
<210>71
<211>225
<212>PRT
<213> human (Homo sapiens)
<400>71
Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu
1 5 10 15
Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln
20 25 30
Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp
35 40 45
Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro
50 55 60
Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe
65 70 75 80
Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln
85 90 95
Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln
100 105 110
Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met
115 120 125
Pro Pro Pro Gly Ala Pro Ser Phe Pro Ser Pro Pro Thr Glu Pro Ser
130 135 140
Ser Glu Val Pro Glu Gln Pro Ser Ala Gln Pro Leu Pro Gly Ser Pro
145 150 155 160
Pro Arg Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu
165 170 175
Val Leu Ser Arg Pro Leu Ser SerAsn Val Ala Thr Leu Gln His Leu
180 185 190
Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr
195 200 205
Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro
210 215 220
Leu
225
<210>72
<211>770
<212>PRT
<213> human (Homo sapiens)
<400>72
Met Ala Gln Trp Asn Gln Leu Gln Gln Leu Asp Thr Arg Tyr Leu Glu
1 5 10 15
Gln Leu His Gln Leu Tyr Ser Asp Ser Phe Pro Met Glu Leu Arg Gln
20 25 30
Phe Leu Ala Pro Trp Ile Glu Ser Gln Asp Trp Ala Tyr Ala Ala Ser
35 40 45
Lys Glu Ser His Ala Thr Leu Val Phe His Asn Leu Leu Gly Glu Ile
50 55 60
Asp Gln Gln Tyr Ser Arg Phe Leu Gln Glu Ser Asn Val Leu Tyr Gln
65 70 75 80
His Asn Leu Arg Arg Ile Lys Gln Phe Leu Gln Ser Arg Tyr Leu Glu
85 90 95
Lys Pro Met Glu Ile Ala Arg Ile Val Ala Arg Cys Leu Trp Glu Glu
100 105 110
Ser Arg Leu Leu Gln Thr Ala Ala Thr Ala Ala Gln Gln Gly Gly Gln
115 120 125
Ala Asn His Pro Thr Ala Ala Val Val Thr Glu Lys Gln Gln Met Leu
130 135 140
Glu Gln His Leu Gln Asp Val Arg Lys Arg Val Gln Asp Leu Glu Gln
145 150 155 160
Lys Met Lys Val Val Glu Asn Leu Gln Asp Asp Phe Asp Phe Asn Tyr
165 170 175
Lys Thr Leu Lys Ser Gln Gly Asp Met Gln Asp Leu Asn Gly Asn Asn
180 185 190
Gln Ser Val Thr Arg Gln Lys Met Gln Gln Leu Glu Gln Met Leu Thr
195 200 205
Ala Leu Asp Gln Met Arg Arg Ser Ile Val Ser Glu Leu Ala Gly Leu
210 215 220
Leu Ser Ala Met Glu Tyr Val Gln Lys Thr Leu Thr Asp Glu Glu Leu
225 230 235 240
Ala Asp Trp Lys Arg Arg Gln Gln Ile Ala Cys Ile Gly Gly Pro Pro
245 250 255
Asn Ile Cys Leu Asp Arg Leu Glu Asn Trp Ile Thr Ser Leu Ala Glu
260 265 270
Ser Gln Leu Gln Thr Arg Gln Gln Ile Lys Lys Leu Glu Glu Leu Gln
275 280 285
Gln Lys Val Ser Tyr Lys Gly Asp Pro Ile Val Gln His Arg Pro Met
290 295 300
Leu Glu Glu Arg Ile Val Glu Leu Phe Arg Asn Leu Met Lys Ser Ala
305 310 315 320
Phe Val Val Glu Arg Gln Pro Cys Met Pro Met His Pro Asp Arg Pro
325 330 335
Leu Val Ile Lys Thr Gly Val Gln Phe Thr Thr Lys Val Arg Leu Leu
340 345 350
Val Lys Phe Pro Glu Leu Asn Tyr Gln Leu Lys Ile Lys Val Cys Ile
355 360 365
Asp Lys Asp Ser Gly Asp Val Ala Ala Leu Arg Gly Ser Arg Lys Phe
370 375 380
Asn Ile Leu Gly Thr Asn Thr Lys Val Met Asn Met Glu Glu Ser Asn
385 390 395 400
Asn Gly Ser Leu Ser Ala Glu Phe Lys His Leu Thr Leu Arg Glu Gln
405 410 415
Arg Cys Gly Asn Gly Gly Arg Ala Asn Cys Asp Ala Ser Leu Ile Val
420 425 430
Thr Glu Glu Leu His Leu Ile Thr Phe Glu Thr Glu Val Tyr His Gln
435 440 445
Gly Leu Lys Ile Asp Leu Glu Thr His Ser Leu Pro Val Val Val Ile
450 455 460
Ser Asn Ile Cys Gln Met Pro Asn Ala Trp Ala Ser Ile Leu Trp Tyr
465 470 475 480
Asn Met LeuThr Asn Asn Pro Lys Asn Val Asn Phe Phe Thr Lys Pro
485 490 495
Pro Ile Gly Thr Trp Asp Gln Val Ala Glu Val Leu Ser Trp Gln Phe
500 505 510
Ser Ser Thr Thr Lys Arg Gly Leu Ser Ile Glu Gln Leu Thr Thr Leu
515 520 525
Ala Glu Lys Leu Leu Gly Pro Gly Val Asn Tyr Ser Gly Cys Gln Ile
530 535 540
Thr Trp Ala Lys Phe Cys Lys Glu Asn Met Ala Gly Lys Gly Phe Ser
545 550 555 560
Phe Trp Val Trp Leu Asp Asn Ile Ile Asp Leu Val Lys Lys Tyr Ile
565 570 575
Leu Ala Leu Trp Asn Glu Gly Tyr Ile Met Gly Phe Ile Ser Lys Glu
580 585 590
Arg Glu Arg Ala Ile Leu Ser Thr Lys Pro Pro Gly Thr Phe Leu Leu
595 600 605
Arg Phe Ser Glu Ser Ser Lys Glu Gly Gly Val Thr Phe Thr Trp Val
610 615 620
Glu Lys Asp Ile Ser Gly Lys Thr Gln Ile Gln Ser Val Glu Pro Tyr
625 630 635 640
Thr Lys Gln Gln Leu Asn Asn Met Ser Phe Ala Glu Ile Ile Met Gly
645 650 655
Tyr Lys Ile Met Asp Ala Thr Asn Ile Leu Val Ser Pro Leu Val Tyr
660 665 670
Leu Tyr Pro Asp Ile Pro Lys Glu Glu Ala Phe Gly Lys Tyr Cys Arg
675 680 685
Pro Glu Ser Gln Glu His Pro Glu Ala Asp Pro Gly Ser Ala Ala Pro
690 695 700
Tyr Leu Lys Thr Lys Phe Ile Cys Val Thr Pro Thr Thr Cys Ser Asn
705 710 715 720
Thr Ile Asp Leu Pro Met Ser Pro Arg Thr Leu Asp Ser Leu Met Gln
725 730 735
Phe Gly Asn Asn Gly Glu Gly Ala Glu Pro Ser Ala Gly Gly Gln Phe
740 745 750
Glu Ser Leu Thr Phe Asp Met Glu Leu Thr Ser Glu Cys Ala Thr Ser
755 760 765
Pro Met
770
<210>73
<211>246
<212>PRT
<213> human (Homo sapiens)
<400>73
Met Glu Leu Val Leu Thr Gln Thr Pro Ser Pro Val Ser Ala Val Val
1 5 10 15
Gly Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Trp Gly
20 25 30
Asn Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg
35 40 45
Leu Leu Met Tyr Tyr Ala Ser Asn Leu Ala Ser Gly Val Ser Ser Arg
50 55 60
Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp
65 70 75 80
Val Gln Cys Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gly Gly Phe Glu
85 90 95
Cys Ser Gly Gly Asp Cys Val Gly Phe Gly Gly Gly Thr Glu Leu Glu
100 105 110
Ile Leu Gly Gly Ser Ser Arg Ser Ser Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Ala
130 135 140
Pro Gly Gly Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu
145 150 155 160
Ser Ser Asp Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
165 170 175
Glu Trp Ile Gly Thr Ile Tyr Gly Ser Ala Gly Thr Tyr Tyr Ala Thr
180 185 190
Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp
195 200 205
Leu Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
210 215 220
Thr Arg Ala Phe Ser Asn Thr Arg Leu Asp Leu Trp Gly Gln Gly Thr
225 230 235 240
Leu Val Thr Ile Ser Ser
245
<210>74
<211>233
<212>PRT
<213> human (Homo sapiens)
<400>74
Met Ser Gln Ser Asn Arg Glu Leu Val Val Asp Phe Leu Ser Tyr Lys
1 5 10 15
Leu Ser Gln Lys Gly Tyr Ser Trp Ser Gln Phe Ser Asp Val Glu Glu
20 25 30
Asn Arg Thr Glu Ala Pro Glu Gly Thr Glu Ser Glu Met Glu Thr Pro
35 40 45
Ser Ala Ile Asn Gly Asn Pro Ser Trp His Leu Ala Asp Ser Pro Ala
50 55 60
Val Asn Gly Ala Thr Gly His Ser Ser Ser Leu Asp Ala Arg Glu Val
65 70 75 80
Ile Pro Met Ala Ala Val Lys Gln Ala Leu Arg Glu Ala Gly Asp Glu
85 90 95
Phe Glu Leu Arg Tyr Arg Arg Ala Phe Ser Asp Leu Thr Ser Gln Leu
100 105 110
His Ile Thr Pro Gly Thr Ala Tyr Gln Ser Phe Glu Gln Val Val Asn
115 120 125
Glu Leu Phe Arg Asp Gly Val Asn Trp Gly Arg Ile Val Ala Phe Phe
130 135 140
Ser Phe Gly Gly Ala Leu Cys Val Glu Ser Val Asp Lys Glu Met Gln
145 150 155 160
Val Leu Val Ser Arg Ile Ala Ala Trp Met Ala Thr Tyr Leu Asn Asp
165 170 175
His Leu Glu Pro Trp Ile Gln Glu Asn Gly Gly Trp Asp Thr Phe Val
180 185 190
Glu Leu Tyr Gly Asn Asn Ala Ala Ala Glu Ser Arg Lys Gly Gln Glu
195 200 205
Arg Phe Asn Arg Trp Phe Leu Thr Gly Met Thr Val Ala Gly Val Val
210 215 220
Leu Leu Gly Ser Leu Phe Ser Arg Lys
225 230
<210>75
<211>312
<212>PRT
<213> human (Homo sapiens)
<400>75
Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala
1 5 10 15
Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu
20 25 30
Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu
35 40 45
Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln
50 55 60
Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Pro Phe
65 70 75 80
Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val
85 90 95
Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln
100 105 110
Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu
115 120 125
Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser
130 135 140
Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly
145 150 155 160
Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val
165 170 175
Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly
180 185 190
Leu Val Asn His Val Pro Leu Glu Gln Met Gln Asn Arg Met Val Ile
195 200 205
Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln
210 215 220
Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala
225 230 235 240
Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe
245 250 255
Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu
260 265 270
Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr
275 280 285
Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln
290 295 300
Thr Met Ser Asn Ser Gly Ile Lys
305 310
<210>76
<211>320
<212>PRT
<213> human (Homo sapiens)
<400>76
Met Pro Met Tyr Gln Val Lys Pro Tyr His Gly Gly Gly Ala Pro Leu
1 5 10 15
Arg Val Glu Leu Pro Thr Cys Met Tyr Arg Leu Pro Asn Val His Gly
20 25 30
Arg Ser Tyr Gly Pro Ala Pro Gly Ala Gly His Val Gln Glu Glu Ser
35 40 45
Asn Leu Ser Leu Gln Ala Leu Glu Ser Arg Gln Asp Asp Ile Leu Lys
50 55 60
Arg Leu Tyr Glu Leu Lys Ala Ala Val Asp Gly Leu Ser Lys Met Ile
65 70 75 80
Gln Thr Pro Asp Ala Asp Leu Asp Val Thr Asn Ile Ile Gln Ala Asp
85 90 95
Glu Pro Thr Thr Leu Thr Thr Asn Ala Leu Asp Leu Asn Ser Val Leu
100 105 110
Gly Lys Asp Tyr Gly Ala Leu Lys Asp Ile Val Ile Asn Ala Asn Pro
115 120 125
Ala Ser Pro Pro Leu Ser Leu Leu Val Leu His Arg Leu Leu Cys Glu
130 135 140
His Phe Arg Val Leu Ser Thr Val His Thr His Ser Ser Val Lys Ser
145 150 155 160
Val Pro Glu Asn Leu Leu Lys Cys Phe Gly Glu Gln Asn Lys Lys Gln
165 170 175
Pro Arg Gln Asp Tyr Gln Leu Gly Phe Thr Leu Ile Trp Lys Asn Val
180 185 190
Pro Lys Thr Gln Met Lys Phe Ser Ile Gln Thr Met Cys Pro Ile Glu
195 200 205
Gly Glu Gly Asn Ile Ala Arg Phe Leu Phe Ser Leu Phe Gly Gln Lys
210 215 220
His Asn Ala Val Asn Ala Thr Leu Ile Asp Ser Trp Val Asp Ile Ala
225 230 235 240
Ile Phe Gln Leu Lys Glu Gly Ser Ser Lys Glu Lys Ala Ala Val Phe
245 250 255
Arg Ser Met Asn Ser Ala Leu Gly Lys Ser Pro Trp Leu Ala Gly Asn
260 265 270
Glu Leu Thr Val Ala Asp Val Val Leu Trp Ser Val Leu Gln Gln Ile
275 280 285
Gly Gly Cys Ser Val Thr Val Pro Ala Asn Val Gln Arg Trp Met Arg
290 295 300
Ser Cys Glu Asn Leu Ala Pro Phe Asn Thr Ala Leu Lys Leu Leu Lys
305 310 315 320
<210>77
<211>233
<212>PRT
<213> human (Homo sapiens)
<400>77
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu
225 230
<210>78
<211>267
<212>PRT
<213> human (Homo sapiens)
<400>78
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys Gly
225 230 235 240
Ser Gly Ser Gly Leu Arg Ser Arg Ala Gln Ala Ser Asn Ser Ala Val
245 250 255
Asp Gly Thr Ala Gly Pro Gly Ser Thr Gly Ser
260 265
<210>79
<211>425
<212>PRT
<213> human (Homo sapiens)
<400>79
Met Ala Asp Lys Glu Ala Ala Phe Asp Asp Ala Val Glu Glu Arg Val
1 5 10 15
Ile Asn Glu Glu Tyr Lys Ile Trp Lys Lys Asn Thr Pro Phe Leu Tyr
20 25 30
Asp Leu Val Met Thr His Ala Leu Glu Trp Pro Ser Leu Thr Ala Gln
35 40 45
Trp Leu Pro Asp Val Thr Arg Pro Glu Gly Lys Asp Phe Ser Ile His
50 55 60
Arg Leu Val Leu Gly Thr His Thr Ser Asp Glu Gln Asn His Leu Val
65 70 75 80
Ile Ala Ser Val Gln Leu Pro Asn Asp Asp Ala Gln Phe Asp Ala Ser
85 90 95
His Tyr Asp Ser Glu Lys Gly Glu Phe Gly Gly Phe Gly Ser Val Ser
100 105 110
Gly Lys Ile Glu Ile Glu Ile Lys Ile Asn His Glu Gly Glu Val Asn
115 120 125
Arg Ala Arg Tyr Met Pro Gln Asn Pro Cys Ile Ile Ala Thr Lys Thr
130135 140
Pro Ser Ser Asp Val Leu Val Phe Asp Tyr Thr Lys His Pro Ser Lys
145 150 155 160
Pro Asp Pro Ser Gly Glu Cys Asn Pro Asp Leu Arg Leu Arg Gly His
165 170 175
Gln Lys Glu Gly Tyr Gly Leu Ser Trp Asn Pro Asn Leu Ser Gly His
180 185 190
Leu Leu Ser Ala Ser Asp Asp His Thr Ile Cys Leu Trp Asp Ile Ser
195 200 205
Ala Val Pro Lys Glu Gly Lys Val Val Asp Ala Lys Thr Ile Phe Thr
210 215 220
Gly His Thr Ala Val Val Glu Asp Val Ser Trp His Leu Leu His Glu
225 230 235 240
Ser Leu Phe Gly Ser Val Ala Asp Asp Gln Lys Leu Met Ile Trp Asp
245 250 255
Thr Arg Ser Asn Asn Thr Ser Lys Pro Ser His Ser Val Asp Ala His
260 265 270
Thr Ala Glu Val Asn Cys Leu Ser Phe Asn Pro Tyr Ser Glu Phe Ile
275 280 285
Leu Ala Thr Gly Ser Ala Asp Lys Thr Val Ala Leu Trp Asp Leu Arg
290 295 300
Asn Leu Lys Leu Lys Leu His Ser Phe Glu Ser His Lys Asp Glu Ile
305 310 315 320
Phe Gln Val Gln Trp Ser Pro His Asn Glu Thr Ile Leu Ala Ser Ser
325 330 335
Gly Thr Asp Arg Arg Leu Asn Val Trp Asp Leu Ser Lys Ile Gly Glu
340 345 350
Glu Gln Ser Pro Glu Asp Ala Glu Asp Gly Pro Pro Glu Leu Leu Phe
355 360 365
Ile His Gly Gly His Thr Ala Lys Ile Ser Asp Phe Ser Trp Asn Pro
370 375 380
Asn Glu Pro Trp Val Ile Cys Ser Val Ser Glu Asp Asn Ile Met Gln
385 390 395 400
Val Trp Gln Met Ala Glu Asn Ile Tyr Asn Asp Glu Asp Pro Glu Gly
405 410 415
Ser Val Asp Pro Glu Gly Gln Gly Ser
420 425
<210>80
<211>375
<212>PRT
<213> human (Homo sapiens)
<400>80
Met Ala Asp His Ser Phe Ser Asp Gly Val Pro Ser Asp Ser Val Glu
1 5 10 15
Ala Ala Lys Asn Ala Ser Asn Thr Glu Lys Leu Thr Asp Gln Val Met
20 25 30
Gln Asn Pro Arg Val Leu Ala Ala Leu Gln Glu Arg Leu Asp Asn Val
35 40 45
Pro His Thr Pro Ser Ser Tyr Ile Glu Thr Leu Pro Lys Ala Val Lys
50 55 60
Arg Arg Ile Asn Ala Leu Lys Gln Leu Gln Val Arg Cys Ala His Ile
65 70 75 80
Glu Ala Lys Phe Tyr Glu Glu Val His Asp Leu Glu Arg Lys Tyr Ala
85 90 95
Ala Leu Tyr Gln Pro Leu Phe Asp Lys Arg Arg Glu Phe Ile Thr Gly
100 105 110
Asp Val Glu Pro Thr Asp Ala Glu Ser Glu Trp His Ser Glu Asn Glu
115 120 125
Glu Glu Glu Lys Leu Ala Gly Asp Met Lys Ser Lys Val Val Val Thr
130 135 140
Glu Lys Ala Ala Ala Thr Ala Glu Glu Pro Asp Pro Lys Gly Ile Pro
145 150 155 160
Glu Phe Trp Phe Thr Ile Phe Arg Asn Val Asp Met Leu Ser Glu Leu
165 170 175
Val Gln Glu Tyr Asp Glu Pro Ile Leu Lys His Leu Gln Asp Ile Lys
180 185 190
Val Lys Phe Ser Asp Pro Gly Gln Pro Met Ser Phe Val Leu Glu Phe
195 200 205
His Phe Glu Pro Asn Asp Tyr Phe Thr Asn Ser Val Leu Thr Lys Thr
210 215 220
Tyr Lys Met Lys Ser Glu Pro Asp Lys Ala Asp Pro Phe Ser Phe Glu
225 230 235 240
Gly Pro Glu Ile Val Asp Cys Asp Gly Cys Thr Ile Asp Trp Lys Lys
245 250 255
Gly Lys Asn Val Thr Val Lys Thr Ile Lys Lys Lys Gln Lys His Lys
260 265 270
Gly Arg Gly Thr Val Arg Thr Ile Thr Lys Gln Val Pro Asn Glu Ser
275 280 285
Phe Phe Asn Phe Phe Asn Pro Leu Lys Ala Ser Gly Asp Gly Glu Ser
290 295 300
Leu Asp Glu Asp Ser Glu Phe Thr Leu Ala Ser Asp Phe Glu Ile Gly
305 310 315 320
His Phe Phe Arg Glu Arg Ile Val Pro Arg Ala Val Leu Tyr Phe Thr
325 330 335
Gly Glu Ala Ile Glu Asp Asp Asp Asn Phe Glu Glu Gly Glu Glu Gly
340 345 350
Glu Glu Glu Glu Leu Glu Gly Asp Glu Glu Gly Glu Asp Glu Asp Asp
355 360 365
Ala Glu Ile Asn Pro Lys Val
370 375
<210>81
<211>1132
<212>PRT
<213> human (Homo sapiens)
<400>81
Met Pro Arg Ala Pro Arg Cys Arg Ala Val Arg Ser Leu Leu Arg Ser
1 5 10 15
His Tyr Arg Glu Val Leu Pro Leu Ala Thr Phe Val Arg Arg Leu Gly
20 25 30
Pro Gln Gly Trp Arg Leu Val Gln Arg Gly Asp Pro Ala Ala Phe Arg
35 40 45
Ala Leu Val Ala Gln Cys Leu Val Cys Val Pro Trp Asp Ala Arg Pro
50 55 60
Pro Pro Ala Ala Pro Ser Phe Arg Gln Val Ser Cys Leu Lys Glu Leu
65 70 75 80
Val Ala Arg Val Leu Gln Arg Leu Cys Glu Arg Gly Ala Lys Asn Val
85 90 95
Leu Ala Phe Gly Phe Ala Leu Leu Asp Gly Ala Arg Gly Gly Pro Pro
100 105 110
Glu Ala Phe Thr Thr Ser Val Arg Ser Tyr Leu Pro Asn Thr Val Thr
115 120 125
Asp Ala Leu Arg Gly Ser Gly Ala Trp Gly Leu Leu Leu Arg Arg Val
130 135 140
Gly Asp Asp Val Leu Val His Leu Leu Ala Arg Cys Ala Leu Phe Val
145 150 155 160
Leu Val Ala Pro Ser Cys Ala Tyr Gln Val Cys Gly Pro Pro Leu Tyr
165 170 175
Gln Leu Gly Ala Ala Thr Gln Ala Arg Pro Pro Pro His Ala Ser Gly
180 185 190
Pro Arg Arg Arg Leu Gly Cys Glu Arg Ala Trp Asn His Ser Val Arg
195 200 205
Glu Ala Gly Val Pro Leu Gly Leu Pro Ala Pro Gly Ala Arg Arg Arg
210 215 220
Gly Gly Ser Ala Ser Arg Ser Leu Pro Leu Pro Lys Arg Pro Arg Arg
225 230 235 240
Gly Ala Ala Pro Glu Pro Glu Arg Thr Pro Val Gly Gln Gly Ser Trp
245 250 255
Ala His Pro Gly Arg ThrArg Gly Pro Ser Asp Arg Gly Phe Cys Val
260 265 270
Val Ser Pro Ala Arg Pro Ala Glu Glu Ala Thr Ser Leu Glu Gly Ala
275 280 285
Leu Ser Gly Thr Arg His Ser His Pro Ser Val Gly Arg Gln His His
290 295 300
Ala Gly Pro Pro Ser Thr Ser Arg Pro Pro Arg Pro Trp Asp Thr Pro
305 310 315 320
Cys Pro Pro Val Tyr Ala Glu Thr Lys His Phe Leu Tyr Ser Ser Gly
325 330 335
Asp Lys Glu Gln Leu Arg Pro Ser Phe Leu Leu Ser Ser Leu Arg Pro
340 345 350
Ser Leu Thr Gly Ala Arg Arg Leu Val Glu Thr Ile Phe Leu Gly Ser
355 360 365
Arg Pro Trp Met Pro Gly Thr Pro Arg Arg Leu Pro Arg Leu Pro Gln
370 375 380
Arg Tyr Trp Gln Met Arg Pro Leu Phe Leu Glu Leu Leu Gly Asn His
385 390 395 400
Ala Gln Cys Pro Tyr Gly Val Leu Leu Lys Thr His Cys Pro Leu Arg
405 410 415
Ala Ala Val Thr Pro Ala Ala Gly Val Cys Ala Arg Glu Lys Pro Gln
420 425 430
Gly Ser Val Ala Ala Pro Glu Glu Glu Asp Thr Asp Pro Arg Arg Leu
435 440 445
Val Gln Leu Leu Arg Gln His Ser Ser Pro Trp Gln Val Tyr Gly Phe
450 455 460
Val Arg Ala Cys Leu Arg Arg Leu Val Pro Pro Gly Leu Trp Gly Ser
465 470 475 480
Arg His Asn Glu Arg Arg Phe Leu Arg Asn Thr Lys Lys Phe Ile Ser
485 490 495
Leu Gly Lys His Ala Lys Leu Ser Leu Gln Glu Leu Thr Trp Lys Met
500 505 510
Ser Val Arg Asp Cys Ala Trp Leu Arg Arg Ser Pro Gly Val Gly Cys
515 520 525
Val Pro Ala Ala Glu His Arg Leu Arg Glu Glu Ile Leu Ala Lys Phe
530535 540
Leu His Trp Leu Met Ser Val Tyr Val Val Glu Leu Leu Arg Ser Phe
545 550 555 560
Phe Tyr Val Thr Glu Thr Thr Phe Gln Lys Asn Arg Leu Phe Phe Tyr
565 570 575
Arg Lys Ser Val Trp Ser Lys Leu Gln Ser Ile Gly Ile Arg Gln His
580 585 590
Leu Lys Arg Val Gln Leu Arg Glu Leu Ser Glu Ala Glu Val Arg Gln
595 600 605
His Arg Glu Ala Arg Pro Ala Leu Leu Thr Ser Arg Leu Arg Phe Ile
610 615 620
Pro Lys Pro Asp Gly Leu Arg Pro Ile Val Asn Met Asp Tyr Val Val
625 630 635 640
Gly Ala Arg Thr Phe Arg Arg Glu Lys Arg Ala Glu Arg Leu Thr Ser
645 650 655
Arg Val Lys Ala Leu Phe Ser Val Leu Asn Tyr Glu Arg Ala Arg Arg
660 665 670
Pro Gly Leu Leu Gly Ala Ser Val Leu Gly Leu Asp Asp Ile His Arg
675 680 685
Ala Trp Arg Thr Phe Val Leu Arg Val Arg Ala Gln Asp Pro Pro Pro
690 695 700
Glu Leu Tyr Phe Val Lys Val Asp Val Thr Gly Ala Tyr Asp Thr Ile
705 710 715 720
Pro Gln Asp Arg Leu Thr Glu Val Ile Ala Ser Ile Ile Lys Pro Gln
725 730 735
Asn Thr Tyr Cys Val Arg Arg Tyr Ala Val Val Gln Lys Ala Ala His
740 745 750
Gly His Val Arg Lys Ala Phe Lys Ser His Val Ser Thr Leu Thr Asp
755 760 765
Leu Gln Pro Tyr Met Arg Gln Phe Val Ala His Leu Gln Glu Thr Ser
770 775 780
Pro Leu Arg Asp Ala Val Val Ile Glu Gln Ser Ser Ser Leu Asn Glu
785 790 795 800
Ala Ser Ser Gly Leu Phe Asp Val Phe Leu Arg Phe Met Cys His His
805 810 815
Ala Val Arg Ile Arg Gly Lys Ser Tyr Val Gln Cys Gln Gly Ile Pro
820 825 830
Gln Gly Ser Ile Leu Ser Thr Leu Leu Cys Ser Leu Cys Tyr Gly Asp
835 840 845
Met Glu Asn Lys Leu Phe Ala Gly Ile Arg Arg Asp Gly Leu Leu Leu
850 855 860
Arg Leu Val Asp Asp Phe Leu Leu Val Thr Pro His Leu Thr His Ala
865 870 875 880
Lys Thr Phe Leu Arg Thr Leu Val Arg Gly Val Pro Glu Tyr Gly Cys
885 890 895
Val Val Asn Leu Arg Lys Thr Val Val Asn Phe Pro Val Glu Asp Glu
900 905 910
Ala Leu Gly Gly Thr Ala Phe Val Gln Met Pro Ala His Gly Leu Phe
915 920 925
Pro Trp Cys Gly Leu Leu Leu Asp Thr Arg Thr Leu Glu Val Gln Ser
930 935 940
Asp Tyr Ser Ser Tyr Ala Arg Thr Ser Ile Arg Ala Ser Leu Thr Phe
945 950 955 960
Asn Arg Gly Phe Lys Ala Gly Arg Asn Met Arg Arg Lys Leu Phe Gly
965 970 975
Val Leu Arg Leu Lys Cys His Ser Leu Phe Leu Asp Leu Gln Val Asn
980 985 990
Ser Leu Gln Thr Val Cys Thr Asn Ile Tyr Lys Ile Leu Leu Leu Gln
995 1000 1005
Ala Tyr Arg Phe His Ala Cys Val Leu Gln Leu Pro Phe His Gln Gln
1010 1015 1020
Val Trp Lys Asn Pro Thr Phe Phe Leu Arg Val Ile Ser Asp Thr Ala
1025 1030 1035 1040
Ser Leu Cys Tyr Ser Ile Leu Lys Ala Lys Asn Ala Gly Met Ser Leu
1045 1050 1055
Gly Ala Lys Gly Ala Ala Gly Pro Leu Pro Ser Glu Ala Val Gln Trp
1060 1065 1070
Leu Cys His Gln Ala Phe Leu Leu Lys Leu Thr Arg His Arg Val Thr
1075 1080 1085
Tyr Val Pro Leu Leu Gly Ser Leu Arg Thr Ala Gln Thr Gln Leu Ser
10901095 1100
Arg Lys Leu Pro Gly Thr Thr Leu Thr Ala Leu Glu Ala Ala Ala Asn
1105 1110 1115 1120
Pro Ala Leu Pro Ser Asp Phe Lys Thr Ile Leu Asp
1125 1130
<210>82
<211>462
<212>PRT
<213> human (Homo sapiens)
<400>82
Met Glu Thr Glu Gln Pro Glu Glu Thr Phe Pro Asn Thr Glu Thr Asn
1 5 10 15
Gly Glu Phe Gly Lys Arg Pro Ala Glu Asp Met Glu Glu Glu Gln Ala
20 25 30
Phe Lys Arg Ser Arg Asn Thr Asp Glu Met Val Glu Leu Arg Ile Leu
35 40 45
Leu Gln Ser Lys Asn Ala Gly Ala Val Ile Gly Lys Gly Gly Lys Asn
50 55 60
Ile Lys Ala Leu Arg Thr Asp Tyr Asn Ala Ser Val Ser Val Pro Asp
65 70 75 80
Ser Ser Gly Pro Glu Arg Ile Leu Ser Ile Ser Ala Asp Ile Glu Thr
85 90 95
Ile Gly Glu Ile Leu Lys Lys Ile Ile Pro Thr Leu Glu Glu Gly Leu
100 105 110
Gln Leu Pro Ser Pro Thr Ala Thr Ser Gln Leu Pro Leu Glu Ser Asp
115 120 125
Ala Val Glu Cys Leu Asn Tyr Gln His Tyr Lys Gly Ser Asp Phe Asp
130 135 140
Cys Glu Leu Arg Leu Leu Ile His Gln Ser Leu Ala Gly Gly Ile Ile
145 150 155 160
Gly Val Lys Gly Ala Lys Ile Lys Glu Leu Arg Glu Asn Thr Gln Thr
165 170 175
Thr Ile Lys Leu Phe Gln Glu Cys Cys Pro His Ser Thr Asp Arg Val
180 185 190
Val Leu Ile Gly Gly Lys Pro Asp Arg Val Val Glu Cys Ile Lys Ile
195 200 205
Ile Leu Asp Leu Ile Ser Glu Ser Pro Ile Lys Gly Arg Ala Gln Pro
210215 220
Tyr Asp Pro Asn Phe Tyr Asp Glu Thr Tyr Asp Tyr Gly Gly Phe Thr
225 230 235 240
Met Met Phe Asp Asp Arg Arg Gly Arg Pro Val Gly Phe Pro Met Arg
245 250 255
Gly Arg Gly Gly Phe Asp Arg Met Pro Pro Gly Arg Gly Gly Arg Pro
260 265 270
Met Pro Pro Ser Arg Arg Asp Tyr Asp Asp Met Ser Pro Arg Arg Gly
275 280 285
Pro Pro Pro Pro Pro Pro Gly Arg Gly Gly Arg Gly Gly Ser Arg Ala
290 295 300
Arg Asn Leu Pro Leu Pro Pro Pro Pro Pro Pro Arg Gly Gly Asp Leu
305 310 315 320
Met Ala Tyr Asp Arg Arg Gly Arg Pro Gly Asp Arg Tyr Asp Gly Met
325 330 335
Val Gly Phe Ser Ala Asp Glu Thr Trp Asp Ser Ala Ile Asp Thr Trp
340 345 350
Ser Pro Ser Glu Trp Gln Met Ala Tyr Glu Pro Gln Gly Gly Ser Gly
355 360 365
Tyr Asp Tyr Ser Tyr Ala Gly Gly Arg Gly Ser Tyr Gly Asp Leu Gly
370 375 380
Gly Pro Ile Ile Thr Thr Gln Val Thr Ile Pro Lys Asp Leu Ala Gly
385 390 395 400
Ser Ile Ile Gly Lys Gly Gly Gln Arg Ile Lys Gln Ile Arg His Glu
405 410 415
Ser Gly Ala Ser Ile Lys Ile Asp Glu Pro Leu Glu Gly Ser Glu Asp
420 425 430
Arg Ile Ile Thr Ile Thr Gly Thr Gln Asp Gln Ile Gln Asn Ala Gln
435 440 445
Tyr Leu Leu Gln Asn Ser Val Lys Gln Tyr Ser Gly Lys Phe
450 455 460
<210>83
<211>745
<212>PRT
<213> human (Homo sapiens)
<400>83
Met Glu Arg Pro Pro Gly Leu Arg Pro Gly Ala Gly Gly Pro Trp Glu
15 10 15
Met Arg Glu Arg Leu Gly Thr Gly Gly Phe Gly Asn Val Cys Leu Tyr
20 25 30
Gln His Arg Glu Leu Asp Leu Lys Ile Ala Ile Lys Ser Cys Arg Leu
35 40 45
Glu Leu Ser Thr Lys Asn Arg Glu Arg Trp Cys His Glu Ile Gln Ile
50 55 60
Met Lys Lys Leu Asn His Ala Asn Val Val Lys Ala Cys Asp Val Pro
65 70 75 80
Glu Glu Leu Asn Ile Leu Ile His Asp Val Pro Leu Leu Ala Met Glu
85 90 95
Tyr Cys Ser Gly Gly Asp Leu Arg Lys Leu Leu Asn Lys Pro Glu Asn
100 105 110
Cys Cys Gly Leu Lys Glu Ser Gln Ile Leu Ser Leu Leu Ser Asp Ile
115 120 125
Gly Ser Gly Ile Arg Tyr Leu His Glu Asn Lys Ile Ile His Arg Asp
130 135 140
Leu Lys Pro Glu Asn Ile Val Leu Gln Asp Val Gly Gly Lys Ile Ile
145 150 155 160
His Lys Ile Ile Asp Leu Gly Tyr Ala Lys Asp Val Asp Gln Gly Ser
165 170 175
Leu Cys Thr Ser Phe Val Gly Thr Leu Gln Tyr Leu Ala Pro Glu Leu
180 185 190
Phe Glu Asn Lys Pro Tyr Thr Ala Thr Val Asp Tyr Trp Ser Phe Gly
195 200 205
Thr Met Val Phe Glu Cys Ile Ala Gly Tyr Arg Pro Phe Leu His His
210 215 220
Leu Gln Pro Phe Thr Trp His Glu Lys Ile Lys Lys Lys Asp Pro Lys
225 230 235 240
Cys Ile Phe Ala Cys Glu Glu Met Ser Gly Glu Val Arg Phe Ser Ser
245 250 255
His Leu Pro Gln Pro Asn Ser Leu Cys Ser Leu Val Val Glu Pro Met
260 265 270
Glu Asn Trp Leu Gln Leu Met Leu Asn Trp Asp Pro Gln Gln Arg Gly
275 280 285
Gly Pro Val Asp LeuThr Leu Lys Gln Pro Arg Cys Phe Val Leu Met
290 295 300
Asp His Ile Leu Asn Leu Lys Ile Val His Ile Leu Asn Met Thr Ser
305 310 315 320
Ala Lys Ile Ile Ser Phe Leu Leu Pro Pro Asp Glu Ser Leu His Ser
325 330 335
Leu Gln Ser Arg Ile Glu Arg Glu Thr Gly Ile Asn Thr Gly Ser Gln
340 345 350
Glu Leu Leu Ser Glu Thr Gly Ile Ser Leu Asp Pro Arg Lys Pro Ala
355 360 365
Ser Gln Cys Val Leu Asp Gly Val Arg Gly Cys Asp Ser Tyr Met Val
370 375 380
Tyr Leu Phe Asp Lys Ser Lys Thr Val Tyr Glu Gly Pro Phe Ala Ser
385 390 395 400
Arg Ser Leu Ser Asp Cys Val Asn Tyr Ile Val Gln Asp Ser Lys Ile
405 410 415
Gln Leu Pro Ile Ile Gln Leu Arg Lys Val Trp Ala Glu Ala Val His
420 425 430
Tyr Val Ser Gly Leu Lys Glu Asp Tyr Ser Arg Leu Phe Gln Gly Gln
435 440 445
Arg Ala Ala Met Leu Ser Leu Leu Arg Tyr Asn Ala Asn Leu Thr Lys
450 455 460
Met Lys Asn Thr Leu Ile Ser Ala Ser Gln Gln Leu Lys Ala Lys Leu
465 470 475 480
Glu Phe Phe His Lys Ser Ile Gln Leu Asp Leu Glu Arg Tyr Ser Glu
485 490 495
Gln Met Thr Tyr Gly Ile Ser Ser Glu Lys Met Leu Lys Ala Trp Lys
500 505 510
Glu Met Glu Glu Lys Ala Ile His Tyr Ala Glu Val Gly Val Ile Gly
515 520 525
Tyr Leu Glu Asp Gln Ile Met Ser Leu His Ala Glu Ile Met Glu Leu
530 535 540
Gln Lys Ser Pro Tyr Gly Arg Arg Gln Gly Asp Leu Met Glu Ser Leu
545 550 555 560
Glu Gln Arg Ala Ile Asp Leu Tyr Lys Gln Leu Lys His Arg Pro Ser
565 570 575
Asp His Ser Tyr Ser Asp Ser Thr Glu Met Val Lys Ile Ile Val His
580 585 590
Thr Val Gln Ser Gln Asp Arg Val Leu Lys Glu Leu Phe Gly His Leu
595 600 605
Ser Lys Leu Leu Gly Cys Lys Gln Lys Ile Ile Asp Leu Leu Pro Lys
610 615 620
Val Glu Val Ala Leu Ser Asn Ile Lys Glu Ala Asp Asn Thr Val Met
625 630 635 640
Phe Met Gln Gly Lys Arg Gln Lys Glu Ile Trp His Leu Leu Lys Ile
645 650 655
Ala Cys Thr Gln Ser Ser Ala Arg Ser Leu Val Gly Ser Ser Leu Glu
660 665 670
Gly Ala Val Thr Pro Gln Thr Ser Ala Trp Leu Pro Pro Thr Ser Ala
675 680 685
Glu His Asp His Ser Leu Ser Cys Val Val Thr Pro Gln Asp Gly Glu
690 695 700
Thr Ser Ala Gln Met Ile Glu Glu Asn Leu Asn Cys Leu Gly His Leu
705 710 715 720
Ser Thr Ile Ile His Glu Ala Asn Glu Glu Gln Gly Asn Ser Met Met
725 730 735
Asn Leu Asp Trp Ser Trp Leu Thr Glu
740 745
<210>84
<211>968
<212>PRT
<213> human (Homo sapiens)
<400>84
Met Ala Glu Asp Asp Pro Tyr Leu Gly Arg Pro Glu Gln Met Phe His
1 5 10 15
Leu Asp Pro Ser Leu Thr His Thr Ile Phe Asn Pro Glu Val Phe Gln
20 25 30
Pro Gln Met Ala Leu Pro Thr Asp Gly Pro Tyr Leu Gln Ile Leu Glu
35 40 45
Gln Pro Lys Gln Arg Gly Phe Arg Phe Arg Tyr Val Cys Glu Gly Pro
50 55 60
Ser His Gly Gly Leu Pro Gly Ala Ser Ser Glu Lys Asn Lys Lys Ser
65 70 7580
Tyr Pro Gln Val Lys Ile Cys Asn Tyr Val Gly Pro Ala Lys Val Ile
85 90 95
Val Gln Leu Val Thr Asn Gly Lys Asn Ile His Leu His Ala His Ser
100 105 110
Leu Val Gly Lys His Cys Glu Asp Gly Ile Cys Thr Val Thr Ala Gly
115 120 125
Pro Lys Asp Met Val Val Gly Phe Ala Asn Leu Gly Ile Leu His Val
130 135 140
Thr Lys Lys Lys Val Phe Glu Thr Leu Glu Ala Arg Met Thr Glu Ala
145 150 155 160
Cys Ile Arg Gly Tyr Asn Pro Gly Leu Leu Val His Pro Asp Leu Ala
165 170 175
Tyr Leu Gln Ala Glu Gly Gly Gly Asp Arg Gln Leu Gly Asp Arg Glu
180 185 190
Lys Glu Leu Ile Arg Gln Ala Ala Leu Gln Gln Thr Lys Glu Met Asp
195 200 205
Leu Ser Val Val Arg Leu Met Phe Thr Ala Phe Leu Pro Asp Ser Thr
210 215 220
Gly Ser Phe Thr Arg Arg Leu Glu Pro Val Val Ser Asp Ala Ile Tyr
225 230 235 240
Asp Ser Lys Ala Pro Asn Ala Ser Asn Leu Lys Ile Val Arg Met Asp
245 250 255
Arg Thr Ala Gly Cys Val Thr Gly Gly Glu Glu Ile Tyr Leu Leu Cys
260 265 270
Asp Lys Val Gln Lys Asp Asp Ile Gln Ile Arg Phe Tyr Glu Glu Glu
275 280 285
Glu Asn Gly Gly Val Trp Glu Gly Phe Gly Asp Phe Ser Pro Thr Asp
290 295 300
Val His Arg Gln Phe Ala Ile Val Phe Lys Thr Pro Lys Tyr Lys Asp
305 310 315 320
Ile Asn Ile Thr Lys Pro Ala Ser Val Phe Val Gln Leu Arg Arg Lys
325 330 335
Ser Asp Leu Glu Thr Ser Glu Pro Lys Pro Phe Leu Tyr Tyr Pro Glu
340 345 350
Ile Lys Asp Lys Glu Glu Val Gln Arg Lys Arg Gln Lys Leu Met Pro
355 360 365
Asn Phe Ser Asp Ser Phe Gly Gly Gly Ser Gly Ala Gly Ala Gly Gly
370 375 380
Gly Gly Met Phe Gly Ser Gly Gly Gly Gly Gly Gly Thr Gly Ser Thr
385 390 395 400
Gly Pro Gly Tyr Ser Phe Pro His Tyr Gly Phe Pro Thr Tyr Gly Gly
405 410 415
Ile Thr Phe His Pro Gly Thr Thr Lys Ser Asn Ala Gly Met Lys His
420 425 430
Gly Thr Met Asp Thr Glu Ser Lys Lys Asp Pro Glu Gly Cys Asp Lys
435 440 445
Ser Asp Asp Lys Asn Thr Val Asn Leu Phe Gly Lys Val Ile Glu Thr
450 455 460
Thr Glu Gln Asp Gln Glu Pro Ser Glu Ala Thr Val Gly Asn Gly Glu
465 470 475 480
Val Thr Leu Thr Tyr Ala Thr Gly Thr Lys Glu Glu Ser Ala Gly Val
485 490 495
Gln Asp Asn Leu Phe Leu Glu Lys Ala Met Gln Leu Ala Lys Arg His
500 505 510
Ala Asn Ala Leu Phe Asp Tyr Ala Val Thr Gly Asp Val Lys Met Leu
515 520 525
Leu Ala Val Gln Arg His Leu Thr Ala Val Gln Asp Glu Asn Gly Asp
530 535 540
Ser Val Leu His Leu Ala Ile Ile His Leu His Ser Gln Leu Val Arg
545 550 555 560
Asp Leu Leu Glu Val Thr Ser Gly Leu Ile Ser Asp Asp Ile Ile Asn
565 570 575
Met Arg Asn Asp Leu Tyr Gln Thr Pro Leu His Leu Ala Val Ile Thr
580 585 590
Lys Gln Glu Asp Val Val Glu Asp Leu Leu Arg Ala Gly Ala Asp Leu
595 600 605
Ser Leu Leu Asp Arg Leu Gly Asn Ser Val Leu His Leu Ala Ala Lys
610 615 620
Glu Gly His Asp Lys Val Leu Ser Ile Leu Leu Lys His Lys Lys Ala
625 630 635640
Ala Leu Leu Leu Asp His Pro Asn Gly Asp Gly Leu Asn Ala Ile His
645 650 655
Leu Ala Met Met Ser Asn Ser Leu Pro Cys Leu Leu Leu Leu Val Ala
660 665 670
Ala Gly Ala Asp Val Asn Ala Gln Glu Gln Lys Ser Gly Arg Thr Ala
675 680 685
Leu His Leu Ala Val Glu His Asp Asn Ile Ser Leu Ala Gly Cys Leu
690 695 700
Leu Leu Glu Gly Asp Ala His Val Asp Ser Thr Thr Tyr Asp Gly Thr
705 710 715 720
Thr Pro Leu His Ile Ala Ala Gly Arg Gly Ser Thr Arg Leu Ala Ala
725 730 735
Leu Leu Lys Ala Ala Gly Ala Asp Pro Leu Val Glu Asn Phe Glu Pro
740 745 750
Leu Tyr Asp Leu Asp Asp Ser Trp Glu Asn Ala Gly Glu Asp Glu Gly
755 760 765
Val Val Pro Gly Thr Thr Pro Leu Asp Met Ala Thr Ser Trp Gln Val
770 775 780
Phe Asp Ile Leu Asn Gly Lys Pro Tyr Glu Pro Glu Phe Thr Ser Asp
785 790 795 800
Asp Leu Leu Ala Gln Gly Asp Met Lys Gln Leu Ala Glu Asp Val Lys
805 810 815
Leu Gln Leu Tyr Lys Leu Leu Glu Ile Pro Asp Pro Asp Lys Asn Trp
820 825 830
Ala Thr Leu Ala Gln Lys Leu Gly Leu Gly Ile Leu Asn Asn Ala Phe
835 840 845
Arg Leu Ser Pro Ala Pro Ser Lys Thr Leu Met Asp Asn Tyr Glu Val
850 855 860
Ser Gly Gly Thr Val Arg Glu Leu Val Glu Ala Leu Arg Gln Met Gly
865 870 875 880
Tyr Thr Glu Ala Ile Glu Val Ile Gln Ala Ala Ser Ser Pro Val Lys
885 890 895
Thr Thr Ser Gln Ala His Ser Leu Pro Leu Ser Pro Ala Ser Thr Arg
900 905 910
Gln Gln Ile Asp Glu Leu Arg Asp Ser Asp Ser Val Cys Asp Ser Gly
915 920 925
Val Glu Thr Ser Phe Arg Lys Leu Ser Phe Thr Glu Ser Leu Thr Ser
930 935 940
Gly Ala Ser Leu Leu Thr Leu Asn Lys Met Pro His Asp Tyr Gly Gln
945 950 955 960
Glu Gly Pro Leu Glu Gly Lys Ile
965