Hibridni proteiniHybrid proteins
Izum se nanaša na hibridne plazminogene aktivatorje, DNA ki kodirajo take hibridne plazminogene aktivatorje, hibridne vek torje, ki vsebujejo take DNA, gostitelje, transformirane s takim hibridnimi vektorji, postopke za pripravo takih hibridnih plazminogenih aktivatorjev DNA, hibridne vektorje in gostitelje, in na farmacevtske sestavke, ki vsebujejo take hibridne plazminogene aktivatorje.The invention relates to hybrid plasminogen activators, DNAs encoding such hybrid plasminogen activators, hybrid vectors containing such DNAs, hosts transformed with such hybrid vectors, methods for preparing such hybrid plasminogen activators DNAs, hybrid vectors and hosts, and pharmaceutical compositions containing such hybrid plasminogen activators.
Krvni strdki so glavni vzrok morbidnosti in umrljivosti ljudi v razvitem svetu.Krvni strdki so sestavljeni iz fibrina, ki se tvori iz svojega topnega predhodnika fibrinogena ob delovanju encima trombina. Vrsta encimov in drugih substanc zagotavlja, da se strdki normalno tvorijo samo, če so in kjer so potrebni za preprečitev izgube krvi.Blood clots are a major cause of morbidity and mortality in humans in the developed world. Blood clots are composed of fibrin, which is formed from its soluble precursor fibrinogen by the action of the enzyme thrombin. A range of enzymes and other substances ensure that clots are normally formed only when and where they are needed to prevent blood loss.
Plazma sesalcev vsebuje encimski sistem, fibrinolitični sistem, ki je zmožen raztapljanja krvnih strdkov. Ena komponenta fibrinolitičnega sistema je skupina encimov, imenovana plazminogeni aktivatorji, ki pretvorijo plazminogen (neaktivna proencimska oblika plazmina) v proteolitičen encim plazmin. Plazmin nato razgradi fibrinsko omrežje strdkov, da nastanejo topni produkti. V primerih, kjer je trombolitična zmožnost telesa nezadostna za odstranitev intravaskularnih trombov, npr.Mammalian plasma contains an enzyme system, the fibrinolytic system, which is capable of dissolving blood clots. One component of the fibrinolytic system is a group of enzymes called plasminogen activators, which convert plasminogen (the inactive proenzyme form of plasmin) into the proteolytic enzyme plasmin. Plasmin then degrades the fibrin network of clots to form soluble products. In cases where the body's thrombolytic capacity is insufficient to remove intravascular thrombi, e.g.
pri pacientih, ki bolujejo na tromboemboliznih ali na postoperativnih komplikacijah, utegne biti nujna uporaba zunanjih trombolitičnih sredstev.In patients suffering from thromboembolic or postoperative complications, the use of external thrombolytic agents may be necessary.
Iz človeških telesnih tekočin ali celic se da izolirati dve vrsti plazminogenih aktivatorjev (v nadaljevanju navedena kot PA): urokinazo ali plazminogeni aktivator tipa urokinaze (v nadaljevanju naveden kot u-PA), serin proteazo, ki se nahaja npr. v človeškem urinu in celicah ledvic ter plazminogeni aktivator tkivne vrste (v nadaljevanju naveden kot t-PA), ki ga proizvajajo endotelne celice in se nahaja v mnogih endokrinih tkivih.Two types of plasminogen activators (hereinafter referred to as PA) can be isolated from human body fluids or cells: urokinase or urokinase-type plasminogen activator (hereinafter referred to as u-PA), a serine protease found, for example, in human urine and kidney cells, and tissue-type plasminogen activator (hereinafter referred to as t-PA), which is produced by endothelial cells and found in many endocrine tissues.
Oba t-PA in u-PA obstajata v dveh molekularnih oblikah: v obliki enojne verige (običajno označena kot sc-t-PA oz. sc-u-PA) in v obliki dveh (tc) verig. Enojna veriga ali oblika proencim se pretvori v obliko dveh verig z· delovanjem proteolitičnih encimov na točno določenih legah v polipeptidnem zaporedju. Rezultirajoči dve verigi obdelanega PA proteina osta neta pritrjeni druga na drugo preko S-S mosta. Karboksi-končni del ali B-veriga posreduje encimsko aktivnost PA-ja, medtem ko amino-končna A-veriga vsebuje regulatorne enote, kot so mesta za vezavo fibrina. Specifična vezava neaktivnega sc-PA na sestavine krvnega strdka, kot je fibrin, ki ji sledi pretvorba v aktivni tc-PA ob katalitičnih količinah proteolitičnih encimov, prisotnih na tem mestu, na koncu daje' učinkovito, položajno specifično zdravilo. t-PA in u-PA, ki ju kodirata dva različna gena, se lahko razlikujeta imunološko in encimsko ter imata različen profil odgovorov na inhibitorje, stimulatorje, in aktivatorje. Tako je samo t-PA močno inhibiran s proteaznim inhibitorjem iz Erytrina latissima (DE-3). Aktivnost t-PA močno stimulirajo fibrin in fibrinski fragmenti, medtem ko je aktivnost u-PA neobčutljiva na stimulacijo s fibrinom in njegovimi fragmenti. Naslednja lastnost za razlikovanje dveh PA encimov je, da ima tc-t-PA visoko afiniteto za fibrin in fibrinske fragmente, medtem ko tc-u-pA'nima znatne fibrinske afinitete.Both t-PA and u-PA exist in two molecular forms: a single-chain form (usually designated as sc-t-PA or sc-u-PA) and a two-chain (tc) form. The single-chain or proenzyme form is converted to a two-chain form by the action of proteolytic enzymes at specific positions in the polypeptide sequence. The resulting two chains of the processed PA protein remain attached to each other via an S-S bridge. The carboxy-terminal portion or B-chain mediates the enzymatic activity of PA, while the amino-terminal A-chain contains regulatory units such as fibrin-binding sites. Specific binding of the inactive sc-PA to blood clot components such as fibrin, followed by conversion to the active tc-PA in the presence of catalytic amounts of proteolytic enzymes present at this site, ultimately results in an effective, site-specific drug. t-PA and u-PA, encoded by two different genes, can be immunologically and enzymatically distinct and have different profiles of responses to inhibitors, stimulators, and activators. Thus, only t-PA is strongly inhibited by the protease inhibitor from Erythrina latissima (DE-3). The activity of t-PA is strongly stimulated by fibrin and fibrin fragments, whereas the activity of u-PA is insensitive to stimulation by fibrin and its fragments. Another feature distinguishing the two PA enzymes is that tc-t-PA has a high affinity for fibrin and fibrin fragments, while tc-u-pA has no significant fibrin affinity.
Glede na nezadovoljivo serumsko stabilnost injiciranih t-PA, nizko afiniteto tc-u-PA za fibrin,in ker menijo, da je fibrinska afiniteta sc-u-PA indirektna, to je, da zahteva dodaten krvni faktor (cf. D.J. Binnema et al., 8th Int. Congress of Fibrinolysis, Vienna, 1986), obstaja nenehna potreba po izboljšanih plazminogenih aktivatorjih z visoko afiniteto do fibrina, bolj ugodnim odgovorom za stimulatorje, zmanjšano inaktivacijo z inhibitorji in daljšo razpoložljivo razpolovno dobo v krvnem obtoku.Given the unsatisfactory serum stability of injected t-PA, the low affinity of tc-u-PA for fibrin, and since the fibrin affinity of sc-u-PA is considered to be indirect, i.e. requiring an additional blood factor (cf. D.J. Binnema et al., 8th Int. Congress of Fibrinolysis, Vienna, 1986), there is a continuing need for improved plasminogen activators with high affinity for fibrin, a more favorable response to stimulators, reduced inactivation by inhibitors, and a longer available half-life in the bloodstream.
Zato je predmet predloženega izuma ustvarjanje novih hibridnih plazminogenih aktivatorjev, ki bodo obdržali koristne lastnosti t-PA in u-PA, medtem ko bodo izgubili nezaželene last4 nosti prvotnih encimov.Therefore, the object of the present invention is to create new hybrid plasminogen activators that will retain the beneficial properties of t-PA and u-PA, while losing the undesirable properties of the original enzymes.
Presenetljivo smo ugotovili, da pri zdravljenju tromboz in drugih stanj, kjer je zaželeno povzročiti fibrinolizo preko plazminogene aktivacije, kažejo eno-verižni hibridni PA proteini boljše biološke lastnosti v primerjavi z eno-verižnimi t-PA in u-PA. Bolj specifično, v primerjavi z nativnimi PA,so potrebne za liziranje krvnih strdkov in vivo manjše množine novih PA molekul v smislu predloženega izuma. Hibridne eno-verižne PA molekule v smislu izuma lahko proizvajamo v velikih količinah preko rekombinantne DNA tehnologije in se bodo šele po injiciranju v paciente pretvorile v dvo-verižno obliko pod vplivom fibrina na mestu liziranja krvnega strdka. Dvo-verižne hibridne PA molekule so opisali v literaturi (Evropska patentna prijava št. 155,387; K.C. Robbins, 8th International Congress of Fibrinolysis, Vienna, 1986), toda bolj ugodne eno-verižne oblike hibridnih PA molekul se ne da proizvajati na nivoju proteinov, kot je navedeno v citirani literaturi, ampak se jih lahko proizvaja le v večjih količinah in v industrijskem obsegu po rekombinantni DNA tehnologiji.Surprisingly, we have found that in the treatment of thrombosis and other conditions where it is desirable to induce fibrinolysis via plasminogen activation, single-chain hybrid PA proteins exhibit superior biological properties compared to single-chain t-PA and u-PA. More specifically, compared to native PA, smaller amounts of the novel PA molecules of the present invention are required to lyse blood clots in vivo. The hybrid single-chain PA molecules of the present invention can be produced in large quantities via recombinant DNA technology and will only be converted to the two-chain form after injection into patients under the influence of fibrin at the site of blood clot lysis. Double-stranded hybrid PA molecules have been described in the literature (European Patent Application No. 155,387; K.C. Robbins, 8th International Congress of Fibrinolysis, Vienna, 1986), but the more favorable single-stranded forms of hybrid PA molecules cannot be produced at the protein level, as stated in the cited literature, but can only be produced in larger quantities and on an industrial scale by recombinant DNA technology.
Torej je nadaljnji predmet predloženega izuma ustvarjanje sredstev in metod za proizvodnjo imenovanih eno-verižnih u-PA/t-PA hibridnih proteinov. Taka sredstva vključujejo DNA,ki kodirajo imenovane u-PA/t-PA hibridne proteine, hibridne vektor je, ki vsebujejo imenovane DNA,in gostitelje, transformirane z imenovanimi hibridnimi vektorji. Ustvarjene so tudi metode za pridobivanje imenovanih eno-verižnih u-PA/t-PA hibridnih proteinov, imenovanih DNA, imenovanih hibridnih vektorjev in imenoThus, a further object of the present invention is to provide means and methods for producing said single-chain u-PA/t-PA hybrid proteins. Such means include DNAs encoding said u-PA/t-PA hybrid proteins, hybrid vectors containing said DNAs, and hosts transformed with said hybrid vectors. Also provided are methods for obtaining said single-chain u-PA/t-PA hybrid proteins, said DNAs, said hybrid vectors, and said
- 5- novanih gostiteljev. Predloženi izum tudi ustvarja cenejši postopek za pridobivanje dvo-verižnih hibridnih PA molekul, ker lahko cepimo eno-verižne produkte rekombinantnih DNA in vitro s primernimi proteolitičnimi encimi, kot je plazmin.- 5- novated hosts. The present invention also creates a less expensive process for obtaining double-stranded hybrid PA molecules because we can cleave single-stranded recombinant DNA products in vitro with suitable proteolytic enzymes such as plasmin.
Podroben opis izumaDetailed description of the invention
Izum se nanaša specifično na eno-verižni hibridni PA, ki ima zaporedje amino kislin sestavljeno iz najmanj dveh podzaporedij, ki ustrezata v enakosti amino kislin in številu podzaporedjem humanega t-PA in humanega u-PA. Podobno kot druge serinske proteaze, vključene v fibrinolitični in koagulacijski sistem krvi, imata u-PA in t-PA velike, nekatalitične segmente, zbrane v verigi A, ki je pritrjena na katalitično regijo (veriga B). Nekatalitično A-verigo t-PA lahko najprej razdelimo v diskretne domene: domena finger, domena rastni faktor in dve strukturi kringle, medtem ko je A veriga u-PA sestavljena iz domene rastni faktor in ene kringle strukture /za referenco glej L. Patthy, Celi 41, 657-663 (1985)/. Katalitično mesto B-verig je sestavljeno iz His, Asp in Ser preostanka na legah 322, 371 in 478 (t-PA) oz. 204, 255 in 356 (u-PA) in je bistveno za fibrinolitično aktivnost.The invention relates specifically to a single-chain hybrid PA having an amino acid sequence consisting of at least two subsequences that correspond in amino acid identity and number to the subsequences of human t-PA and human u-PA. Similar to other serine proteases involved in the fibrinolytic and coagulation systems of blood, u-PA and t-PA have large, non-catalytic segments assembled in the A chain, which is attached to a catalytic region (B chain). The non-catalytic A chain of t-PA can first be divided into discrete domains: a finger domain, a growth factor domain and two kringle structures, while the A chain of u-PA consists of a growth factor domain and one kringle structure /for reference see L. Patthy, Cells 41, 657-663 (1985)/. The catalytic site of the B-chains consists of His, Asp and Ser residues at positions 322, 371 and 478 (t-PA) and 204, 255 and 356 (u-PA), respectively, and is essential for fibrinolytic activity.
Proteinska domena je strukturna in/ali funkcionalna enota v celotni strukturi vsega proteina. Npr. v t-PA A verige so štiri domene (finger , rastni faktor in dve kringle domeni) razvrščene zaporedno. Meje domen so najbolje definirane z legami ekson-intron zvez v odgovarjajočem DNA zaporedju /L. Patthy, zgoraj). Vendar pa je bila iz smotrnih razlogov definirana minimalna velikost vsake domene z zaporedjem amino kislin med prvim in zadnjim cisteinskim preostankom znotraj vsake domene, ki se verjetno vključujejo v tvorbo S-S mosta. Amino kisline pred in za temi cisteinskimi preostanki sosednjih domen so definirane kot vezna zaporedja (J). Lege zvez eksonintron (glej zgoraj) so v teh J regijah.A protein domain is a structural and/or functional unit in the overall structure of a protein. For example, in the t-PA A chain, four domains (finger, growth factor, and two kringle domains) are arranged in a sequential manner. The domain boundaries are best defined by the positions of the exon-intron junctions in the corresponding DNA sequence (L. Patthy, supra). However, for practical reasons, the minimum size of each domain has been defined by the sequence of amino acids between the first and last cysteine residues within each domain that are likely to be involved in the formation of the S-S bridge. The amino acids before and after these cysteine residues of adjacent domains are defined as the junctional sequences (J). The positions of the exon-intron junctions (see above) are in these J regions.
Tako lahko predstavimo eno-verižni t-PA z naslednjo formulo: T - F- J] - G - J2 - K1 - - K2 - - TPAB, kjer T pomeni N-končni del, ki obsega amino kisline 1 do 5,Thus, single-chain t-PA can be represented by the following formula: T - F- J] - G - J2 - K1 - - K2 - - TPAB , where T represents the N-terminal part, comprising amino acids 1 to 5,
F je domena finger, ki obsega amino kisline 6 do 43, G je domena rastni faktor, ki obsega amino kisline 51 do 84, K< je kringle 1 struktura, ki obsega amino kisline 92 do 173, je B kringle 2 struktura, ki obsega amino kisline 180 do 261, TPA je območje katalitične serin proteaze, ki obsega amino kisline 307 do 527 in (amino kisline 44 do 50), J? (amino kisline 85 do 91), (amino kisline 174 do 179) in amino kisline 262 do 306) so vezna zaporedja, ki povezujejo segmente domen.F is a finger domain comprising amino acids 6 to 43, G is a growth factor domain comprising amino acids 51 to 84, K< is a kringle 1 structure comprising amino acids 92 to 173, B is a kringle 2 structure comprising amino acids 180 to 261, TPA is a serine protease catalytic region comprising amino acids 307 to 527 and (amino acids 44 to 50), J? (amino acids 85 to 91), (amino acids 174 to 179) and amino acids 262 to 306) are linker sequences connecting the domain segments.
Eno-verižni u-PA lahko predstavimo z naslednjo formulo:Single-chain u-PA can be represented by the following formula:
T’ - u - j5 - K - j6 - UPAB kjer T’ pomeni N-končni del, ki obsega amino kisline 1 do 12,T' - u - j5 - K - j6 - UPAB where T' represents the N-terminal part comprising amino acids 1 to 12,
U je domena rastni faktor, ki obsega amino kisline 13 do 42,U is a growth factor domain comprising amino acids 13 to 42,
K je struktura kringle, ki obsega amino kisline 50 do 131, UPA je območje katalitične serin proteaze, ki obsega amino kisline 189 do 411 in (amino kisline 43 do 49) in (amino kisline 132 do 188) so vezna zaporedja, ki povezujejo segmente domen.K is a kringle structure spanning amino acids 50 to 131, UPA is the catalytic serine protease region spanning amino acids 189 to 411, and (amino acids 43 to 49) and (amino acids 132 to 188) are linker sequences connecting the domain segments.
Vsako od veznih zaporedij in Jg vsebuje aktivacijsko (presnovno) mesto in še N-končni, cisteinski preostanek, ki je vključen v S-S most v katalitičnem (B-veriga) območju.Each of the binding sequences and Jg contains an activation (metabolic) site and an N-terminal, cysteine residue that is involved in an S-S bridge in the catalytic (B-chain) region.
Presenetljivo smo ugotovili, da eno-verižni hibridniSurprisingly, we found that single-stranded hybrid
PA-ji, ki vsebujejo katalitično območje serin proteaze enega B B od PA (TPA ali UPA ), pritrjeno na zaporedje amino kislin, ki vsebuje vse ali diskretne domene A-verige drugih PA ali diskretne domene obeh PA, kažejo koristne farmakološke lastnosti.PAs containing the serine protease catalytic domain of one B chain of PAs (TPA or UPA) attached to an amino acid sequence containing all or discrete A-chain domains of other PAs or discrete domains of both PAs exhibit beneficial pharmacological properties.
Izum se torej nanaša na eno-verižni hibridni PA, ki obsega zaporedje amino kislin, ki vsebuje vse ali diskretne domene A-verige humanega u-PA ali diskretne domene A-verige humanega u-PA in humanega t-PA,vezane zaporedno na katalitično območje humanega t-PA (TPA ) in na eno-verižni hibridni PA, ki obsega zaporedje amino kislin, ki vsebuje vse ali diskretne domene A-verige humanega t-PA ali diskretne domene A-verige humanega t-PA in u-PA,vezane zaporedno na katalitično območje humanega u-PA (UPA ). Pri prednostni izvedbi vsebujejo hibridni BThe invention therefore relates to a single-chain hybrid PA comprising an amino acid sequence containing all or discrete domains of the A-chain of human u-PA or discrete domains of the A-chain of human u-PA and human t-PA linked sequentially to the catalytic region of human t-PA (TPA) and to a single-chain hybrid PA comprising an amino acid sequence containing all or discrete domains of the A-chain of human t-PA or discrete domains of the A-chain of human t-PA and u-PA linked sequentially to the catalytic region of human u-PA (UPA). In a preferred embodiment, the hybrid B
PA v smislu izuma katalitično območje humanega u-PA (UPA ).The PA in the context of the invention is the catalytic region of human u-PA (UPA).
Izum se zlasti nanaša na eno-verižne PA, ki obsegajo zaporedje amino kislin, izbrano iz skupine, ki sestoji iz zaporedja amino kislin, ki vsebuje vse domene A-verige humanega t-PA, zaporedja amino kislin, ki vsebuje diskretne domene Averige humanega t-PA, kot so domene finger ali kringle, zlasti domena kringle 2 humanega t-PA in zaporedja amino kislin, ki vsebuje dve, tri ali štiri domene A-verige humanega t-PA in/ali humanega u-PA, zlasti dve ali tri domene humanega t-PA ali dve ali tri domene humanega u-PA in humanega t-PA, kot so domene finger, rastni faktor in kringle 2 humanega t-PA, domeni finger in kringle 2 humanega t-PA ali domeni rastni faktor u-PA in kringle 2 t-PA, katerih zaporedje amino kislin je vezano zaporedno na katalitično območje humanega u-PA in na eno-verižni PA, ki obsega zaporedje amino kislin, ki vsebuje domeni rastni faktor u-PA in kringle 2 t-PA, katerih zaporedje amino kislin je vezano zaporedno na katalitično območje humanega t-PA.The invention particularly relates to single-chain PAs comprising an amino acid sequence selected from the group consisting of an amino acid sequence containing all the A-chain domains of human t-PA, an amino acid sequence containing discrete A-chain domains of human t-PA, such as finger or kringle domains, in particular the kringle 2 domain of human t-PA, and an amino acid sequence containing two, three or four A-chain domains of human t-PA and/or human u-PA, in particular two or three domains of human t-PA or two or three domains of human u-PA and human t-PA, such as the finger, growth factor and kringle 2 domains of human t-PA, the finger and kringle 2 domains of human t-PA or the growth factor u-PA and kringle 2 domains of t-PA, the amino acid sequence of which is linked sequentially to the catalytic region of human u-PA and to a single-chain PA comprising an amino acid sequence containing the growth factor domains u-PA and kringle 2 t-PA, whose amino acid sequence is linked sequentially to the catalytic region of human t-PA.
Prednostno se prične zaporedje amino kislin hibridnega PA z N-končnim zaporedjem t-PA (T, amino kisline 1 do 5) ali u-PA (Τ’, amino kisline 1 do 12)?ali se prične s katerim koli veznim zaporedjem, ki je naravno N-končno vezano na prvo domeno hibridnega PA,ali s fragmentom takega veznega zaporedja, katerega fragment ima prednostno najmanj pet amino kislinskih preostankov.Preferably, the amino acid sequence of the hybrid PA begins with the N-terminal sequence of t-PA (T, amino acids 1 to 5) or u-PA (Τ', amino acids 1 to 12)? or begins with any linker sequence that is naturally N-terminally linked to the first domain of the hybrid PA, or with a fragment of such a linker sequence, which fragment preferably has at least five amino acid residues.
V hibridnih PA v smislu izuma so domene na A-verigi povezane preko naravnih veznih zaporedij (npr. J^, J2, inIn hybrid PAs of the invention, the domains on the A-chain are connected via natural linker sequences (e.g., J^, J2 , and
J._), zlitih veznih zaporedij, ali hibridnih veznih zaporedij b ali njihovih fragmentov.J._), fused binding sequences, or hybrid binding sequences b or fragments thereof.
8a8a
Domene A-verige hibridnih PA v smislu izuma so povezane z B-verigo serin proteaznega območja (TPAB ali UPAB) z veznim zaporedjem, izbranim iz skupine, ki sestoji iz veznega zaporedja «J^, ki povezuje A_verigo z B-verigo v humanem t-PA, veznega zaporedja J^, ki povezuje A-verigo z B-verigo v humanem u-PA in hibridnega zaporedja, sestavljenega iz podzaporedij imenovanih veznih zaporedij, pri čemer imenovano vezno zaporedje vključuje aktivacijsko mesto, ki ga je zmožen razcepiti plazmin in še Nkončni cisteinski preostanek, ki lahko sodeluje v mostu Žvepložveplo do katalitičnega območja B-verige, kjer ima vezno zaporedje prednostno najmanj štirideset pa tja do 60 amino kislinskih preostankov.The A-chain domains of hybrid PAs according to the invention are linked to the B-chain of the serine protease region (TPAB or UPAB ) by a linker sequence selected from the group consisting of the linker sequence «J^, which connects the A_chain to the B-chain in human t-PA, the linker sequence J^, which connects the A-chain to the B-chain in human u-PA and a hybrid sequence consisting of subsequences of said linker sequences, said linker sequence including an activation site capable of being cleaved by plasmin and an N-terminal cysteine residue capable of participating in a Sulfur-Sulfur bridge to the catalytic region of the B-chain, where the linker sequence preferably has at least forty and up to 60 amino acid residues.
Najbolj prednostna je povezava domen na legi, ki je definirana z zvezami ekson-intron na odgovarjajoči DNA. Zveza A-verige z B-verigo je predvsem prednostna na aktivacijskem mestu.The most preferred location for domain association is defined by the exon-intron junctions on the corresponding DNA. The A-chain to B-chain association is particularly preferred at the activation site.
Tako je prva domena vezana na drugo domeno z veznim zaporedjem, ki se naravno pojavlja v C-končnem delu prve do mene, z veznim zaporedjem, ki se naravno pojavlja v N-končnem delu prve domene, z veznim zaporedjem, ki se naravno pojavlja na N-končnem delu druge domene, z zlitim veznim zaporedjem, ki je sestavljeno iz navedenih veznih zaporedij, ali z njihovimi fragmenti.Thus, the first domain is linked to the second domain by a linker sequence that naturally occurs in the C-terminal part of the first domain, by a linker sequence that naturally occurs in the N-terminal part of the first domain, by a linker sequence that naturally occurs in the N-terminal part of the second domain, by a fusion linker sequence consisting of said linker sequences, or by fragments thereof.
- 9 Zlasti se izum nanaša na eno-verižni hibridni plazminogeni aktivator,izbran iz skupine, ki sestoji iz takega hibridnega plazminogenega aktivatorja, ki obsega A-verigo u-PA ali A-verigo,sestavijeno na značilen način iz domen rastni faktor u-PA in kringle 2 t-PA, vezanih zaporedno na katalitično območje (B-veriga) t-PA ter na hibridni plazminogeni aktivator, ki obsega A-verigo t-PA, A-verigo,sestavijeno na značilen način iz domene finger t-PA, A-verigo, sestavljeno na značilen način iz domen rastni faktor u-PA in kringle 2 t-PA, Averigo, sestavljeno na značilen način iz domen finger t-PA in kringle 2, ali A-verigo,sestavijeno na značilen način iz domen finger t-PA, rastni faktor in kringle 2 imenovane Averige, ki je vezana zaporedno na katalitično območje (B-veriga) u-PA, pri čemer je A-veriga vezana na B-verigo preko veznega zaporedja, ki vsebuje aktivacijsko mesto in cisteinski preostanek, ki je sposoben tvorjenja S-S vezi do B-verige.- 9 In particular, the invention relates to a single-chain hybrid plasminogen activator selected from the group consisting of such a hybrid plasminogen activator comprising an A-chain of u-PA or an A-chain composed in a characteristic manner of the u-PA growth factor and kringle 2 t-PA domains linked in series to the catalytic region (B-chain) of t-PA and to a hybrid plasminogen activator comprising an A-chain of t-PA, an A-chain composed in a characteristic manner of the t-PA finger domain, an A-chain composed in a characteristic manner of the u-PA growth factor and kringle 2 t-PA domains, an Averigo composed in a characteristic manner of the t-PA finger and kringle 2 domains, or an A-chain composed in a characteristic manner of the t-PA finger, growth factor and kringle 2 domains called Averigo, which is linked in a characteristic manner to the catalytic region (B-chain) of u-PA, wherein The A-chain is linked to the B-chain via a linker sequence that contains an activation site and a cysteine residue capable of forming an S-S bond to the B-chain.
Zlasti se izum nanaša tudi na eno-verižni hibridni plazminogeni aktivator, ki obsega A-verigo, sestavljeno na značilen način iz domene kringle 2 t-PA. vezano na katalitično območje (B-veriga) u-PA na aktivacijskem mestu.In particular, the invention also relates to a single-chain hybrid plasminogen activator comprising an A-chain, composed in a characteristic manner of the kringle 2 domain of t-PA, linked to the catalytic region (B-chain) of u-PA at the activation site.
Posebno prednosten je eno-verižni hibridni plazminogeni aktivator, izbran iz skupine, ki sestoji iz takega hibridnega plazminogenega aktivatorja, ki obsega A-verigo, sestavljeno na značilen način iz domene rastni faktor u-PA in domene kringle 2 t-PA, vezano zaporedno na katalitično območje (B -veriga) t-PA in na hibridni plazminogeni aktivator, ki obsega A-verigo, sestavljeno na značilen način iz domene kringle 2 t-PA alvi domen finger in kringle 2 t-PA, vezano zaporedno na katalitično območje (B--veriga) u-PA, pri čemer je povezav.a med domeno (domenami) A-verige in B-verigo ob aktivacijskem mestu.Particularly preferred is a single-chain hybrid plasminogen activator selected from the group consisting of such a hybrid plasminogen activator comprising an A-chain typically composed of a u-PA growth factor domain and a kringle 2 t-PA domain linked sequentially to the catalytic region (B-chain) of t-PA and a hybrid plasminogen activator comprising an A-chain typically composed of a kringle 2 t-PA domainand a finger and kringle 2 t-PA domain linked sequentially to the catalytic region (B-chain) of u-PA, wherein the linkage between the A-chain domain(s) and the B-chain is at the activation site.
Prednostni hibridni PAfji v smislu izuma so:Preferred hybrid PAFs according to the invention are:
UPA^TPAB(BC) - [uPA(l-158)-tPA(276-527)],UPA^TPAB (BC) - [uPA(1-158)-tPA(276-527)],
UPA^TPAB(BR) = [uPA(l-131)-tPA(263-527)],UPA^TPAB (BR) = [uPA(1-131)-tPA(263-527)],
TPAAUPAB(BC) - [tPA(l-275)-uPA(l59-411)J,TPAA UPAB (BC) - [tPA(l-275)-uPA(l59-411)J,
TPA^UPAB(BR) = [tPA(l-262)-uPA(132-411)],TPA^UPAB (BR) = [tPA(1-262)-uPA(132-411)],
UK2UPAB(BR) = [uPA(l-44)-tPA(l76-261)-uPA(134-411)],UK2 UPAB (BR) = [uPA(l-44)-tPA(l76-261)-uPA(134-411)],
FUPAB(BC) » [tPA(l-49)-tPA(262-275)-uPA(159-411)],FUPAB (BC) » [tPA(l-49)-tPA(262-275)-uPA(159-411)],
FUPAB(BR) - (tPA(l-49)-uPA(134-411)],FUPAB (BR) - (tPA(1-49)-uPA(134-411)],
FK2UPAB(BC) « (tPA(l-49)-tPA(176-275)-uPA(159-411) ],FK2 UPAB (BC) « (tPA(1-49)-tPA(176-275)-uPA(159-411) ],
FK2UPAB(BR) = [tPA(l-49)-tPA(176-262)-uPA(132-4ll) ],FK2 UPAB (BR) = [tPA(l-49)-tPA(176-262)-uPA(132-4ll) ],
UK2TPA(BC) = (tPA(l-44)-tPA(176-527)],UK2 TPA(BC) = (tPA(1-44)-tPA(176-527)],
K2UPAC(BC) = (tPA(l-3)-tPA(176-275)-uPA(159-411)],K2 UPAC (BC) = (tPA(1-3)-tPA(176-275)-uPA(159-411)],
FGK2UPAB(BC) = [tPA(l-86)-tPA(176-275)-uPA(159-411) ] inFGK2 UPAB (BC) = [tPA(1-86)-tPA(176-275)-uPA(159-411) ] and
FGK2UPAB(BR) » [tPA(l-86)-tPA(176-262)-uPA( 132-411)],’FGK2 UPAB (BR) » [tPA(l-86)-tPA(176-262)-uPA( 132-411)],'
A A pri katerih je UPA A-veriga u-PA, TPA je v A-verigi t-PA,A A in which UPA is the A-chain of u-PA, TPA is in the A-chain of t-PA,
UPA je B-veriga u-PA, TPA je B-veriga t-PA, U se nanaša na domeno rastni faktor u-PA, K? se nanaša na domeno kringle 2 t-PA, F se nanaša na domeno finger t-PA, G se nanaša na domeno rastni faktor t-PA, (BC) označuje, da je zveza med domeno (domenami) A-verige in B-verigo ob aktivacijskem mestuUPA is the B-chain of u-PA, TPA is the B-chain of t-PA, U refers to the u-PA growth factor domain, K? refers to the kringle 2 domain of t-PA, F refers to the finger domain of t-PA, G refers to the growth factor domain of t-PA, (BC) indicates that the junction between the A-chain and B-chain domain(s) is at the activation site
1 in (BR) označuje, da je (so) domena (e) A-verige vezane na B-verigo preko veznega zaporedja, naravno pritrjenega na B-verigo, ki vključuje aktivacijsko mesto,in nadalje N-končno, cisteinski preostanek, ki je vključen v S-S most do B-verige. Števila se nanašajo na zaporedja aminokislinj vzeta iz u-PA oz. t-PA. Npr. UK2UPAB(BR) = /uPA(1-44)-tPA(176-261)-uPA(134-411)/ označuje eno-verižni hibridni plazminogeni aktivator, ki sestoji iz amino kislin 1-44 (domena rastni faktor, U), u-PA in amino kislin 176-261 (domena kringle 2, K2) t-PA, vezanih na linearen način na amino kisline 134-411 (B-veriga, UPA ) u-PA.1 and (BR) indicates that the domain(s) of the A-chain are linked to the B-chain via a linker sequence naturally attached to the B-chain, which includes the activation site, and further N-terminally, a cysteine residue that is involved in the SS bridge to the B-chain. The numbers refer to the amino acid sequences taken from u-PA or t-PA. For example, UK2 UPAB (BR) = /uPA(1-44)-tPA(176-261)-uPA(134-411)/ indicates a single-chain hybrid plasminogen activator consisting of amino acids 1-44 (growth factor domain, U) of u-PA and amino acids 176-261 (kringle domain 2, K2 ) of t-PA, linked in a linear fashion to amino acids 134-411 (B-chain, UPA ) of u-PA.
Zlasti prednostni so hibridni plazminogeni aktivatorji TPAAUPAB(BC), FUPAB(BC), FGK2UPAB(BC) in zlasti UK2TPAB(BC), FK2UPAB(BC) in K2UPAB(BC).Particularly preferred are the hybrid plasminogen activators TPAA UPAB (BC), FUPAB (BC), FGK2UPAB (BC) and especially UK2TPAB (BC), FK2UPAB (BC) and K2UPAB (BC).
Izum se nadalje nanaša na mutante hibridnih PA-jev v smislu izuma, pri katerih je (so) najmanj eno, prednostno vsa N-glikoz.ilacijska mesta modificirana tako, da glikozilacija ne more potekati na tem (teh) mestu (mestih). Dobro je znano, da je prvi pogoj za N-vezano glikozilacijo v celicah sesalcev prisotnost tripeptidnega zaporedja -Asn-L-Ser-(ali Thr)-, pri čemer je Asn akceptor in je L lahko katerakoli od 20 genetično kodiranih amino kislin razen prolina ali asparginove kisline, ki preprečujeta glikozilacijo. V t-PA molekuli so tri mesta za N-glikozidno vezavo (števila se nanašajo na lego Asn v zaporedju amino kislin t-PA, primerjaj sl. 1, spremljajočih risb):The invention further relates to mutants of hybrid PAs according to the invention, in which at least one, preferably all N-glycosylation sites are modified in such a way that glycosylation cannot take place at this (these) site(s). It is well known that the first condition for N-linked glycosylation in mammalian cells is the presence of the tripeptide sequence -Asn-L-Ser-(or Thr)-, where Asn is the acceptor and L can be any of the 20 genetically encoded amino acids except proline or aspartic acid, which prevent glycosylation. There are three sites for N-glycosidic linkage in the t-PA molecule (the numbers refer to the position of Asn in the amino acid sequence of t-PA, compare Fig. 1, accompanying drawings):
-Asn1 1 ^-Ser-Ser- (prisotno v kringle 1), Asn1^^-Gly-Ser 448 (prisotno v kringle 2) in Asn -Arg-Thr (prisotno v t-PA B-verige). Edino N-vezano glikozilacijsko mesto u-PA je v B-veri12-Asn1 1 ^-Ser-Ser- (present in kringle 1), Asn1 ^^-Gly-Ser 448 (present in kringle 2) and Asn -Arg-Thr (present in t-PA B-chain). The only N-linked glycosylation site of u-PA is in the B-chain12
302 gi (Asn -Ser-Thr, prim. sl. 3)· Jasno je, da hibridni PA, ki obsegajo t-PA kringle 1, t-PA kringle 2, B-verigo t-PA in/ ali B-verigo u-PA, tudi vključujejo posamezna N-vezana glikozilaeijska mesta.302 gi (Asn -Ser-Thr, cf. Fig. 3)· It is clear that hybrid PAs comprising t-PA kringle 1, t-PA kringle 2, t-PA B-chain and/or u-PA B-chain also include individual N-linked glycosylation sites.
Zato, da preprečimo glikozilacijo na posameznih (enem ali več) N-glikozilacijskih mestih, se morajo spremeniti zaporedja v tripeptidih, spoznana kot signali za N-glikozilacijo. Zamenjava Asn in/ali Ser (ali Thr) preostankov v zgornjih tripeptidnih zaporedjih s katerokoli drugo amino kislino, bi uničila tvorbo glikozidnih vezi na teh mestih. Zaradi pridLadnosti modifikacija N-glikozilacijskih mest ni izvedena na proteinskem nivoju. Namesto tega je ugodno modificirati gen, ki kodira hibridni PA na tak način, da se po ekspresiji imenovanega modificiranega gena v gostitelju proizvede mutanta hibridnega PA, v kateri je eno ali več N-glikozilacijskih mest spremenjenih na tak način, da glikozilacija ne more potekati na teh mestih. Prednostno je modificirati vsa N-glikozilaeijska mesta, ki se pojavljajo v hibrid nih PA v smislu izuma.In order to prevent glycosylation at individual (one or more) N-glycosylation sites, sequences in the tripeptides known as signals for N-glycosylation must be modified. Replacing Asn and/or Ser (or Thr) residues in the above tripeptide sequences with any other amino acid would destroy the formation of glycosidic bonds at these sites. For convenience, the modification of the N-glycosylation sites is not carried out at the protein level. Instead, it is advantageous to modify the gene encoding the hybrid PA in such a way that, upon expression of said modified gene in a host, a mutant of the hybrid PA is produced in which one or more N-glycosylation sites are modified in such a way that glycosylation cannot occur at these sites. It is preferable to modify all N-glycosylation sites occurring in the hybrid PAs of the invention.
Predvsem je asparagin zamenjan z valinom,levcinom, izolevcinom, alaninom ali zlasti glutaminom,in serin ali treonin z valinom, metioninom ali zlasti alaninom.In particular, asparagine is replaced by valine, leucine, isoleucine, alanine or especially glutamine, and serine or threonine by valine, methionine or especially alanine.
Predvsem so prednostni modificirani hibridni PA:In particular, modified hybrid PAs are preferred:
FUPAB(Gln302)(BC) = [ tPA(l-49)-tPA(262-275)-uPA(159-3Ol,FUPAB (Gln302)(BC) = [ tPA(1-49)-tPA(262-275)-uPA(159-3Ol,
Gin, 303-411)],Gin, 303-411)],
FKz(Alal86)UPAB(Gln302)(BC) = [tPA(l-49)-tPA(176-185, Ala, 187-275)uPA(159-301, Gin, 303-411)],FKwith (Alal86)UPAB (Gln302)(BC) = [tPA(1-49)-tPA(176-185, Ala, 187-275)uPA(159-301, Gln, 303-411)],
UK2(Alal86)TPAB(Ala450)(BC) - [uPA(l-44)-tPA(176-185, Ala, 187-449,UK2 (Alal86)TPAB (Ala450)(BC) - [uPA(1-44)-tPA(176-185, Ala, 187-449,
Ala, 451-527)],Ala, 451-527)],
K2(Alal86)UPAB(Gln302)(BC) = (tPA(l-3)-tPA(176-185, Ala, 187-275)uPA(159-301, Gin, 303-411)],K2 (Alal86)UPAB (Gln302)(BC) = (tPA(1-3)-tPA(176-185, Ala, 187-275)uPA(159-301, Gln, 303-411)],
FGK2(Alal86)UPAB(Gln3O2)(BC) «= [tPA(l-86)-tPA( 1 76-185, Ala, 187-275)ruPA(159-3Ol, Gin, 303-411)], in nadaljeFGK2 (Alal86)UPAB (Gln3O2)(BC) «= [tPA(l-86)-tPA( 1 76-185, Ala, 187-275)r uPA(159-3Ol, Gin, 303-411)], and further
FK2UPAB(Gln302)(BC) = [ tPA(l-49)-tPA(176-275)-uPA(159-301, Gin,FK2 UPAB (Gln302)(BC) = [ tPA(1-49)-tPA(176-275)-uPA(159-301, Gin,
303-411)],303-411)],
K2UPAB(Gln302)(BC) = [tPA(l-3)-tPA(l76-275)-uPA( 159-301, Gin,K2 UPAB (Gln302)(BC) = [tPA(1-3)-tPA(176-275)-uPA( 159-301, Gin,
303-411)],303-411)],
UK2TPAB(Ala450)(BC) = [uPA(l-44)-tPA(176-449, Ala, 451-527)], in FGK2UPAB(Gln302)(BC) = (tPA(l-86)-tPA(l76-275)-uPA( 159-301, Gin,UK2 TPAB (Ala450)(BC) = [uPA(l-44)-tPA(176-449, Ala, 451-527)], and FGK2 UPAB (Gln302)(BC) = (tPA(l-86)-tPA(l76-275)-uPA( 159-301, Gln302)(BC)
303-411)].303-411)].
Hibridne PA in njihove mutante v smislu izuma lahko pripravimo po rekombinantni DNA tehniki, ki obsega npr. gojenje transformiranega gostitelja, ki izraža hibridni PA protein ali njegovo mutanto pod, pogoji,ki dopuščajo njihovo ekspresijo in izolacijo hibridnega PA proteina oz. mutante hibridnega PA proteina. Bolj specifično, zaželene spojine proizvajamo s:Hybrid PAs and their mutants according to the invention can be prepared by recombinant DNA techniques, which comprise, for example, culturing a transformed host expressing the hybrid PA protein or a mutant thereof under conditions that allow for their expression and isolation of the hybrid PA protein or mutant of the hybrid PA protein. More specifically, the desired compounds are produced by:
a) pridobivanjem DNA, ki kodira hibridni PA protein in njegovo mutanto, ali kemično sintetiziranje take DNAa) obtaining DNA encoding the hybrid PA protein and its mutant, or chemically synthesizing such DNA
b) vključevanje DNA v primeren ekspresijski vektorb) incorporating the DNA into a suitable expression vector
c) prenašanje dobljenega hibridnega vektorja v sprejemnega gostiteljac) transferring the resulting hybrid vector into a recipient host
d) selekcioniranje transformiranega gostitelja od netransformiranih gostiteljev, npr. z gojenjem pod pogoji, pod katerimi preživi samo transformirani gostiteljd) selecting the transformed host from non-transformed hosts, e.g. by growing under conditions under which only the transformed host survives
e) gojenje transformiranega gostitelja pod pogoji, ki dopuščajo ekspresijo hibridnega PA proteina in ,Λe) cultivating the transformed host under conditions that allow expression of the hybrid PA protein and ,Λ
f) izoliranje hibridnega PA proteina ali njegove mutante.f) isolating the hybrid PA protein or a mutant thereof.
|7f _|7f _
Stopnje, ki so vključene v pripravo hibridnih PA proteinov po rekombinantni DNA tehniki, bomo podrobno opisali v nadaljevanju.The steps involved in the preparation of hybrid PA proteins by recombinant DNA technology will be described in detail below.
DNA, ki kodirajo hibridne PA proteineDNA encoding hybrid PA proteins
Izum se nanaša na DNA, ki imajo zaporedje, ki kodira hibridni PA,ki je sestavljen iz najmanj dveh podzaporedij, ki ustrezata po identiteti in številu amino kislin podzaporedjema humanega u-PA in humanega t-PA, ali ki kodira njegovo mutanto. Zlasti se izum nanaša na DNA, ki imajo zaporedje, ki kodira vsakega od hibridnih PA proteinov in njihovih mutant, še omenjene kot prednostne.The invention relates to DNAs having a sequence encoding a hybrid PA consisting of at least two subsequences corresponding in identity and amino acid number to the subsequences of human u-PA and human t-PA, or encoding a mutant thereof. In particular, the invention relates to DNAs having a sequence encoding each of the hybrid PA proteins and their mutants, also mentioned as preferred.
Prednostno imajo DNA v smislu izuma priležna zaporedja na svojih koncih. Zlasti ta priležna zaporedja vključujejo primerna restrikcijska mesta,ki omogočajo integriranje DNA v primerne vektorjePreferably, the DNAs of the invention have flanking sequences at their ends. In particular, these flanking sequences include suitable restriction sites which allow integration of the DNA into suitable vectors.
Nadalje DNA v smislu izuma vključujejo signalno zapored je za u-PA ali t-PA,pritrjeno na prvi kodon zaporedja, ki kodira zrel hibridni PA. Če se izražajo v celicah kvasovk, lahko DNA v smislu izuma vsebujejo alternativno signalno zaporedje kvasa, kot je signalno zaporedje, naravno povezano z uporabljenim promotorjem kvasovk, zlasti PH05 ali invertazno signalno zaporedje.Furthermore, the DNAs of the invention include a signal sequence for u-PA or t-PA attached to the first codon of the sequence encoding the mature hybrid PA. When expressed in yeast cells, the DNAs of the invention may contain an alternative yeast signal sequence, such as a signal sequence naturally associated with the yeast promoter used, in particular PH05, or an invertase signal sequence.
Prednostno so zaporedja nukleotidov v podzaporedjih DNA identična z zaporedji nukleotidov v cDNA u-PA oz. cDNA t-PA. Vendar pa se zaradi degeneracije genetičnega koda lahko zaporedja nukleotidov razlikujejo, če ostane rezultirajoče zaporedje amino kislin nespremenjeno. V DNA, ki kodirajo mutanto hibridnega PA, je najmanj en kodon, ki kodira amino kislino, bistveno za N-glikozilacijo v hibridnem PA proteinu, nadomeščen z drugim kodonom, ki kodira drugo amino kislino, ki uniči spoznavno mesto za N-glikozilacijo.Preferably, the nucleotide sequences in the DNA subsequences are identical to the nucleotide sequences in the u-PA cDNA or t-PA cDNA, respectively. However, due to the degeneracy of the genetic code, the nucleotide sequences may differ, provided that the resulting amino acid sequence remains unchanged. In DNA encoding a mutant hybrid PA, at least one codon encoding an amino acid essential for N-glycosylation in the hybrid PA protein is replaced by a second codon encoding a second amino acid that destroys the recognition site for N-glycosylation.
Zaporedja nukleotidov za u-PA cDNA in t-PA cDNA so znana /cf. W.E. Holmes et. al., Biotechnology 3, 923-929 (1985); D. Penniea et al., Nature 301, 214-221 (1983)/. Nadalje so bila dognana vsa zaporedja nukleotidov v genomu za gene u-PA in t-PA, vključno z vsemi introni in eksoni /cf. A. Riccio et al., Nucl. Acids Res. 13, 2759-2771 (1985); S.J. Friezner-Degen et al.,The nucleotide sequences for u-PA cDNA and t-PA cDNA are known /cf. W.E. Holmes et. al., Biotechnology 3, 923-929 (1985); D. Penniea et al., Nature 301, 214-221 (1983)/. Furthermore, all nucleotide sequences in the genome for the u-PA and t-PA genes, including all introns and exons, have been determined /cf. A. Riccio et al., Nucl. Acids Res. 13, 2759-2771 (1985); S.J. Friezner-Degen et al.,
J. Biol. Chem. 261, 6972-6985 (1986)/.J. Biol. Chem. 261, 6972-6985 (1986)/.
Pri poznavanju zaporedij cDNA in genomske DNA za u-PA in t-PA lahko izdelamo DNA v smislu izuma po metodah, znanih v stroki. Metode za izdelavo teh DNA vključujejo kemično sintetiziranje DNA ali pripravljanje fragmentov, ki kodirajo podzaporedja polinukleotidov za u-PA cDNA in t-PA cDNA in njihovo ponovno vezavo v vnaprej določeno ureditev po izbiri, ki vključuje eno ali več, kot so dve ali tri stopnje mutacije.Knowing the cDNA and genomic DNA sequences for u-PA and t-PA, DNAs of the invention can be produced by methods known in the art. Methods for producing these DNAs include chemically synthesizing DNA or preparing fragments encoding polynucleotide subsequences for u-PA cDNA and t-PA cDNA and re-ligating them in a predetermined arrangement, optionally involving one or more, such as two or three, stages of mutation.
DNA, ki kodirajo mutante hibridnih PA,se v smislu izuma lahko izdelajo po metodah,znanih v stroki. Metode za izdelovanje teh DNA vključujejo izrezovanje dela DNA, ki vsebuje kodon za nezaželeni amino kislinski preostanek7iz gena za parentalni hibridni PA in njegovo nadomestitev z DNA segmentom, v čemer je bil imenovani kodon nadomeščen z deoksiribonukleotidnim tripletom, ki kodira želeni amino kislinski preostanek, ali opravljanje deoksiribonukleotidne zamenjave s pomočjo na /nešto usmerjene mutageneze.DNAs encoding mutants of hybrid PAs according to the invention can be produced by methods known in the art. Methods for producing these DNAs include excising a portion of DNA containing the codon for the undesired amino acid residue7 from the gene for the parental hybrid PA and replacing it with a DNA segment in which said codon has been replaced by a deoxyribonucleotide triplet encoding the desired amino acid residue, or performing the deoxyribonucleotide substitution by site-directed mutagenesis.
-ί-6’~-ί-6’~
Kemična sinteza DNA je dobro znana v stroki in uporablja običajne tehnike. Primerne tehnike je zbral S.A. Narang /Tetrahedron 39, 3 (1983)/. Zlasti metode, opisane v Evropski patentni prijavi št. 146,785 se lahko uporabljajo in so vključene kot reference.Chemical synthesis of DNA is well known in the art and uses conventional techniques. Suitable techniques have been reviewed by S.A. Narang /Tetrahedron 39, 3 (1983)/. In particular, the methods described in European Patent Application No. 146,785 may be used and are incorporated by reference.
Drug pristop za sintezo DNA v smislu izuma sestoji iz izrezanja primernih restrikcijskih fragmentov, ki kodirajo polinukleotidna podzaporedja za u-PA in t-PA, iz u-PA cDNA in t-PA c-DNA (ali genomske u-PA DNA ali t-PA DNA) in uporabe teh fragmentov za pripravo strukturnega gena za celotni hibridni PA. Uporabljamo lahko dve strategiji. Pri obeh strategijah moramo skrbeti, da pride do zlitja fragmentov na mestih med domenami, zato,da se slednje ohranijo nepoškodovane. Prva strategija uporablja primerna restrikcijska mesta. Ko je primerno restrikcijsko mesto na razpolago na vnaprej določenem veznem mestu (mestih) v obeh u-PA in t-PA DNA, se DNA prebavijo z odgovarjajočo restrikcijsko endonukleazo ter se fragmenti povežejo s topimi ali odprtimi konci (brez nasprotnih baz) (odvisno od izbrane restrikcijske endonukleaze). Druga možnost je, da se lahko vpeljejo uporabna restrikcijska mesta, npr. z na mesto usmerjeno mutagenezo /cf. M.J. Zoller et al., Methods Enzymol 100, 468 (1983)/, in pazimo, da mutirana DNA ne rezultira v spremenjenem zaporedju amino kislin. Posebno prednostna so tista naravna ali umetno vpeljana restrikcijska mesta, ki ločijo DNA, ki kodirajo A- in B-verige ali DNA, ki kodirajo diskretne domene, ki jih vsebujejo A-verige. Na ta način lahko proizvajamo hibridne DNA, katere kodirajo hibridne PA, ki imajo zaželeno povezavo med domenami A-verige in katalitičnim, serinproteaznim območjem.Another approach for DNA synthesis according to the invention consists of excising suitable restriction fragments encoding polynucleotide subsequences for u-PA and t-PA from u-PA cDNA and t-PA cDNA (or genomic u-PA DNA or t-PA DNA) and using these fragments to prepare the structural gene for the entire hybrid PA. Two strategies can be used. In both strategies, care must be taken to ensure that the fragments are fused at sites between the domains, in order to keep the latter intact. The first strategy uses suitable restriction sites. Once a suitable restriction site is available at a predetermined binding site(s) in both u-PA and t-PA DNA, the DNA is digested with the appropriate restriction endonuclease and the fragments are ligated with blunt or open ends (without opposing bases) (depending on the restriction endonuclease chosen). Alternatively, useful restriction sites can be introduced, e.g. by site-directed mutagenesis /cf. M.J. Zoller et al., Methods Enzymol 100, 468 (1983)/, and care is taken that the mutated DNA does not result in an altered amino acid sequence. Particularly preferred are those natural or artificially introduced restriction sites that separate DNAs encoding the A- and B-chains or DNAs encoding discrete domains contained within the A-chains. In this way, hybrid DNAs can be produced that encode hybrid PAs that have the desired linkage between the A-chain domains and the catalytic, serine protease region.
Druga strategija izhaja iz hipoteze, da so meje domen najbolje definirane z lego ekson-intron povezav v DNA—genomih/cf. L. Patthy, Celi 41, 657-663 (1985)/, to je lege v cDNA, kjer so se introni povezali. Ker te lege redko sovpadajo z restrikci jskimi mesti, je prisvojena shema, ki ji lahko sledimo za vsako novo konstrukcijo: v prvi stopnji se primerni restrikcijski fragmenti, ki kodirajo specifično domeno (e), ter vsebujejo tudi dodatna DNA zaporedja, razen pričakovane točke zlitja (do več sto baznih parov),povežejo in subklonirajo v bakteriofagu ml3- V drugi stopnji se prekomerna DNA zaporedja razformirajo z in vitro mutagenezo (Zoller et al., zgoraj). Ta postopek dovoljuje zlitja v natančno določenem okviru na vsaki vnaprej določeni legi nukleotida in je zato prednosten.The second strategy is based on the hypothesis that domain boundaries are best defined by the location of exon-intron junctions in DNA genomes/cf. L. Patthy, Cells 41, 657-663 (1985)/, i.e. the locations in the cDNA where introns have been spliced. Since these locations rarely coincide with restriction sites, a scheme has been adopted that can be followed for any new construction: in the first step, suitable restriction fragments encoding the specific domain(s) and also containing additional DNA sequences, apart from the expected fusion site (up to several hundred base pairs), are ligated and subcloned in bacteriophage ml3. In the second step, the excess DNA sequences are eliminated by in vitro mutagenesis (Zoller et al., supra). This procedure allows for in-frame fusions at each predetermined nucleotide position and is therefore preferred.
Za pripravo mutante hibridnih PA lahko izvedeno izrezanje dela zrele hibridne DNA z uporabo restrikcijskih encimov. Prvi pogoj te metode je možnost, da spremenimo primerna restrik cijska mesta v bližini kodona. Majhen restrikcijski fragment, ki vsebuje kodon za nezaželeno amino kislino,odstranimo s cepljenjem z endonukleazo. Pripravimo odgovarjajoče zaporedje dvoj no vlaknate DNA,npr. s pomočjo kemične sinteze, v kateri uporab ljamo triplete,ki kodirajo zaželeno amino kislino, DNA fragment povežemo v prikladni orientaciji na ostanek velikega fragmenta, da dobimo zaporedje dvojno vlaknate DNA, ki kodira mutanto hibrida. Iz udobnosti in zaradi lažjega dela s hibridnim genom, je ta prikladno vsebovan v večjem DNA segmentu, kar ustvarimo s primernimi povezovalci, ki dovoljujejo vključitev in kloniranje segmenta v klonirnem vektorju.To prepare a mutant hybrid PA, a portion of the mature hybrid DNA can be excised using restriction enzymes. The first requirement of this method is the ability to modify suitable restriction sites near the codon. A small restriction fragment containing the codon for the unwanted amino acid is removed by endonuclease cleavage. The corresponding double-stranded DNA sequence is prepared, e.g. by chemical synthesis using triplets encoding the desired amino acid, and the DNA fragment is ligated in a suitable orientation to the remainder of the large fragment to obtain the double-stranded DNA sequence encoding the hybrid mutant. For convenience and ease of handling, the hybrid gene is conveniently contained within a larger DNA segment, which is created with suitable linkers that allow the segment to be inserted and cloned in a cloning vector.
Τδ V prednostni izvedbi predloženega izuma izvedemo pripra vo DNA, ki kodirajo mutanto hibridnega PA,z na mesto usmerjeno mutagenezo. Ta metoda je postopek mutageneze in vitro, s katero se lahko spremeni določeno mesto znotraj regije klonirane DNA /prim. pregl. članke M.J. Zoller, M. Smith, Methods Enzymol. 100, 468 1983); D. Botsbein, D. Shortle, Science 229, 1193 (1985)/.. Mutagenezo lahko izvedemo ali na celotnem genu hibridnega PA ali na njegovem funkcionalnem delu, ki vsebuje kodon za nezaželeno amino kislino (e). Po mutagenezi povežemo mutirani funkcionalni del z drugimi deli hibridnega PA, da dobimo mutanto hibridnega PA.Τδ In a preferred embodiment of the present invention, DNA encoding a mutant of the hybrid PA is prepared by site-directed mutagenesis. This method is an in vitro mutagenesis procedure that can alter a specific site within a region of cloned DNA /cf. review articles by M.J. Zoller, M. Smith, Methods Enzymol. 100, 468 1983); D. Botsbein, D. Shortle, Science 229, 1193 (1985)/.. Mutagenesis can be performed either on the entire gene of the hybrid PA or on its functional part containing the codon for the undesired amino acid (e). After mutagenesis, the mutated functional part is linked to other parts of the hybrid PA to obtain a mutant of the hybrid PA.
Metoda mutiranja hibridnega PA gena ali njegovega funkcionalnega dela je značilna v tem, da se eno-vlaknat gen ali eno-vlaknata DNA, ki vsebuje PA gen ali njegov del,hibridizira z oligodeoksiribonukleotidnim pnimerjem, ki je komplementaren regiji hibridnega gena, ki naj bi bil mutiran, razen pri slabih parjenjih (parjenju), ki usmerjajo (usmerja) mutacijo? hibridiziran oligodeoksiribonukleotid uporabljamo kot primer, da začne sintezo komplementarnega DNA vlakna, nastala (delno) dvojnovlaknata DNA se transformira v sprejemni soj mikroorganizma, le-tega gojimo inJ selekcioniramo transformante, ki vsebujejo DNA, z modificiranim (mutiranim) genom za hibridni PA.The method of mutating a hybrid PA gene or its functional part is characterized in that a single-stranded gene or single-stranded DNA containing a PA gene or part thereof is hybridized with an oligodeoxyribonucleotide primer that is complementary to the region of the hybrid gene to be mutated, except for poor pairings that direct the mutation? The hybridized oligodeoxyribonucleotide is used as a primer to initiate the synthesis of a complementary DNA strand, the resulting (partially) double-stranded DNA is transformed into a recipient strain of the microorganism, the latter is cultivated, and transformants containing DNA with the modified (mutated) gene for the hybrid PA are selected.
Hibridni vektorji, ki vsebujejo DNA hibridnega PAHybrid vectors containing hybrid PA DNA
Izum se nanaša na hibridne vektorje, ki vsebujejo DNA, ki kodira hibridni PA, kateri je sestavljen iz najmanj dveh podzaporedij, ki ustrezajo po identiteti in številu amino kislin podzaporedjem humanega u-PA in humanega t-PA, ali kodirajo njegovo mutanto,in na postopke za njihovo pripravo.The invention relates to hybrid vectors containing DNA encoding a hybrid PA consisting of at least two subsequences corresponding in identity and number of amino acids to the subsequences of human u-PA and human t-PA, or encoding a mutant thereof, and to methods for their preparation.
Vektor izberemo v odvisnosti od celic gostitelja, planiranih za transformacijo. V principu so primerni vsi vektorji, ki se podvojujejo in izražajo želeni polipeptidni gen v smislu izuma v izbranem gostitelju. Primeri ustreznih gostiteljev so evkarionti,ki so brez ali z malo restrikcijskimi encimi ali modificiranimi encimi, kot kvasovke, npr. Saccharomyces cerevisiae, npr. S. cervisiae GRF18 in nadalje celice sesalcev, zlasti navedene humane ali živalske celične linije, npr. mieloma celice, humani embrionalni pljučni fibroblasti L-132, COS celice, LTK celice, humane, rakaste melanoma Bowes celice, HeLa celice, z virusom SV-40 transformirane ledvične celice afriške zelene opice COS-7 ali celice ovarija kitajskega hrčka (CHO) in njihove variante. Prednostne kot gostiteljski mikroorganizmi,so gornje celice sesalcev in soji Saccharomyces cerevisiae, npr. S. cerevisiae GRF18,The vector is selected depending on the host cells planned for transformation. In principle, all vectors that replicate and express the desired polypeptide gene according to the invention in the selected host are suitable. Examples of suitable hosts are eukaryotes that are free of or with few restriction enzymes or modified enzymes, such as yeasts, e.g. Saccharomyces cerevisiae, e.g. S. cervisiae GRF18, and furthermore mammalian cells, in particular the aforementioned human or animal cell lines, e.g. myeloma cells, human embryonic lung fibroblasts L-132, COS cells, LTK cells, human melanoma Bowes cells, HeLa cells, SV-40 virus-transformed African green monkey kidney cells COS-7 or Chinese hamster ovary (CHO) cells and variants thereof. Preferred as host microorganisms are the above mammalian cells and the soybean Saccharomyces cerevisiae, e.g. S. cerevisiae GRF18,
a. Vektorji za uporabo v kvasua. Vectors for use in yeast
Vektorji, ki so primerni za podvojevanje in ekspresijo v kvasu,vsebujejo podvojevalni začetek kvasa in selektivni genetični označevalec za kvas. Hibridni vektorji, ki vsebujejo podvojevalni začetek kvasa, npr. kromosomski segment (lokus), ki se neodvisno podvojuje, ostanejo zunaj kromosoma v celici kvasa po transformaciji in se neodvisno podvojujejo. Nadalje se lahko uporabljajo hibridni vektorji, ki vsebujejo zaporedja, homologna 2u plazmidni DNA kvasa. Taki hibridni vektorji se bodo integrirali z rekombinacijo v 2μ plazmide, ki že obstaja20 jo v celici, ali se neodvisno podvojevali. 2p zaporedja so zlasti primerna za plazmide, ki imajo visoko frekvenco transformacije in dovoljujejo visoka števila kopij.Vectors suitable for replication and expression in yeast contain a yeast origin of replication and a yeast selectable genetic marker. Hybrid vectors containing a yeast origin of replication, e.g. a chromosome segment (locus) that replicates independently, remain outside the chromosome in the yeast cell after transformation and replicate independently. Hybrid vectors containing sequences homologous to yeast 2u plasmid DNA can also be used. Such hybrid vectors will either integrate by recombination into 2μ plasmids already existing in the cell or replicate independently. 2p sequences are particularly suitable for plasmids that have a high transformation frequency and permit high copy numbers.
Primerni označevalni geni za kvas so zlasti tisti, ki podeljujejo antibiotično odpornost gostitelju, ali v primeru auksotrofnih mutant kvasa,geni, ki popravljajo poškodbe gostitelja. Odgovarjajoči geni podelijo npr. odpornost proti antibiotiku G418 ali oskrbijo zmožnost fotosinteze v auksotrofni mutanti kvasa, npr. URA3, LEV2, HIS3 ali TRP1 gen. Hibridni vektorji kvasa nadalje prednostno vsebujejo podvojevalni začetek in označevalni gen za bakterijskega gostitelja, zlasti E.coli, tako da konstrukcija in kloniranje hibridnih vektorjev in njihovih intermediatov lahko poteka v bakterijskem gostitelju.Suitable marker genes for yeast are in particular those which confer antibiotic resistance to the host, or in the case of auxotrophic yeast mutants, genes which repair host damage. Corresponding genes confer e.g. resistance to the antibiotic G418 or provide the ability to photosynthesize in auxotrophic yeast mutants, e.g. the URA3, LEV2, HIS3 or TRP1 gene. The yeast hybrid vectors further preferably contain an origin of replication and a marker gene for the bacterial host, in particular E. coli, so that the construction and cloning of the hybrid vectors and their intermediates can take place in the bacterial host.
Ekspresijska kontrolna zaporedja, ki so primerna za ekspresijo v kvasu,so npr. izmed dobro izraženih genov kvasa. Tako lahko uporabljamo promotorje TRP1 gena, ADHI ali ADHII gena, gene kisle fosfataze (PHO3 ali PHO5), gen izocitokroma ali promotor za gene glikolize, kot je promotor genov enolaze, gliceraldehid-3-fosfat dehidrogenaze (GAPDH), 3-fosfoglicerat kinaze (PGK), heksokinaze, piruvat dekarboksilaze, fosfofruktokinaze, glukoza-6-fosfat izomeraze, 3-fosfoglicerat mutaze, piruvat kinaze, trioza fosfat izomeraze, fosfoglukoza izomeraze, invertaze in glukokinaze. Prednostni vektorji predloženega izuma vsebujejo promotorje s prepisovalno kontrolo, npr. promotorje PHO5 in ADHII genov, ki se lahko vključijo ali izključijo pri spreminjanju rastnih pogojev. Npr. PHO5 promotor lahko zaustavimo ali sprostimo izključno s povečanjem ali znižanjem koncentracije anorganskega fosfata v mediju. Prednostno hibrid ni vektorji kvasa v smislu predloženega izuma obsegajo tudi 3’ priležno zaporedje gena kvasa, ki vsebuje primeren signal za prenehanje prepisovanja in poliadenilacijo. Ustrezna 3’ priležna zaporedja so npr. tista, ki so v genu naravno vezana na uporabljeni promotor, kot je 3 ’ priležno zapored je v genu PHO5 kvasa.Expression control sequences suitable for expression in yeast are, for example, from well-expressed yeast genes. Thus, promoters of the TRP1 gene, the ADHI or ADHII gene, the acid phosphatase gene (PHO3 or PHO5), the isocytochrome gene or promoters for glycolysis genes, such as the promoter of the enolase gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 3-phosphoglycerate kinase (PGK), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, invertase and glucokinase, can be used. Preferred vectors of the present invention contain promoters with transcriptional control, for example, the promoters of the PHO5 and ADHII genes, which can be switched on or off when the growth conditions are changed. For example, The PHO5 promoter can be silenced or released solely by increasing or decreasing the concentration of inorganic phosphate in the medium. Preferably, the yeast hybrid vectors of the present invention also comprise a 3' flanking sequence of the yeast gene containing a suitable signal for termination of transcription and polyadenylation. Suitable 3' flanking sequences are, for example, those that are naturally associated in the gene with the promoter used, such as the 3' flanking sequence in the yeast PHO5 gene.
b. Vektorji za uporabo v celicah sesalcevb. Vectors for use in mammalian cells
Vektorji za podvojevanje in ekspresijo v celicah sesalcev so pogosto oskrbljeni z DNA virusnega izvora, npr. iz opičjega virusa 40 (SV40), virusa Rousovega sarkoma (RSV), adenovirusa 2, volovskega papiloma virusa (BPV), mutante papovavirusa (BK (BKV) ali mišjega ali humanega citomegalovirusa (MCMV oz. HCMV).Vectors for replication and expression in mammalian cells are often provided with DNA of viral origin, e.g. from simian virus 40 (SV40), Rous sarcoma virus (RSV), adenovirus 2, bovine papillomavirus (BPV), mutant papovavirus (BK (BKV) or murine or human cytomegalovirus (MCMV or HCMV).
Ekspresijska kontrolna zaporedja, ki so primerna za upo rabo v celicah sesalcev, vključujejo, med drugim, zgodnje in pozne promotorje SV40, večji pozni promotor adeno virusa, promotor metalotioneinskega gena glodalcev in področje povečevalnik-promotor v mišjem ali humanem citomegalo virusnem večjem nenadno-zgodnjem genu, območje povečevalnega promotorja humanega imunoglobulina, promotor humanega ©//-globina, po izbiri v kombinaciji s SV40 povečevalnikom,in promotorje, izvedene iz toplotno šokiranih genov.Expression control sequences suitable for use in mammalian cells include, but are not limited to, the SV40 early and late promoters, the adenovirus major late promoter, the rodent metallothionein gene promoter and the enhancer-promoter region in the mouse or human cytomegalovirus major sudden-early gene, the human immunoglobulin enhancer promoter region, the human β-globin promoter, optionally in combination with the SV40 enhancer, and promoters derived from heat-shocked genes.
Primerna označevalna gena za celice sesalcev sta npr. neo in ble gena t ranspozona Tn5, ki podeljujeta odpornost proti antibiotiku G418 oz. antibiotiku vrste bleomicin, gen E.coli, higromicin-B odpornost, gen za dihidrofolat reduktazo (dhfr) iz celic sesalcev ali E.coli, ki spremeni fenotip DHFR- celic v DHFR+ celice in/ali podeljujejo odpornost protimethotrexatu in gen timidin kinaze virusa Herpes simplex, ki napravi TKcelice fenotipično TK+ celice.Suitable marker genes for mammalian cells are, for example, the neo and ble genes of the Tn5 transposon, which confer resistance to the antibiotic G418 or the antibiotic bleomycin, the E. coli gene, hygromycin-B resistance, the gene for dihydrofolate reductase (dhfr) from mammalian cells or E. coli, which alters the DHFR phenotype.-cells in DHFR+cells and/or confer resistance to methotrexate and the Herpes simplex virus thymidine kinase gene, which makes TKcells phenotypically TK+cells.
Prednostno vsebujejo hibridni vektorji za celice sesalcev 3’ neprevajani del gena sesalcev, ki vsebuje signale za primerni zaključek prepisovanja in poliadenilacijo, kot npr. 3’ priležni del gena za /S-globin. Ugodnejše, priležni na področje, ki kodira polipeptid, vključujejo enega ali več nativnih intronov, ki imajo primerne signale za vezanje na svojih koncih. Taki dodatki se smatrajo za potrebne, ker cDNA in prokariontske DNA, kot zgornji selekcijski geni, pogosto nimajo takih prepisovalnih in procesnih signalov.Preferably, hybrid vectors for mammalian cells contain a 3' untranslated portion of a mammalian gene that contains signals for appropriate transcription termination and polyadenylation, such as, for example, the 3' flanking portion of the β-globin gene. More preferably, flanking the polypeptide coding region, they include one or more native introns that have appropriate splicing signals at their ends. Such additions are considered necessary because cDNAs and prokaryotic DNAs, such as the above selection genes, often lack such transcription and processing signals.
Za razmnoževanje v E.coli prednostno vsebujejo vektorji začetek prepisovanja in antibiotično odporen gen. Začetek podvojevanja pri sesalcih lahko zagotovimo ali z vključitvijo eukariontskega začetka v konstrukcijo vektor ja »tistega, ki izhaja iz SV40 ali drugega virusnega izvora, ali ga lahko zagotovimo s kromosomom celice gostiteljice po integraciji vektorja v kromosom celice gostiteljice.For replication in E. coli, vectors preferably contain an origin of transcription and an antibiotic resistance gene. Initiation of replication in mammals can be provided either by incorporating a eukaryotic origin into the vector construction, such as that derived from SV40 or other viral origin, or it can be provided by the host cell chromosome after integration of the vector into the host cell chromosome.
Prednostni hibridni vektorji, ki jih uporabljamo v celicah sesalcev, obsegajo cDNA hibridnega PA ali mutante hibridnega PA, delovno priležno obdanega na navzgor obrnjeni strani s citomegalovirusnim glodalčnim nenadno-zgodnjim genskim povečevalnikom-promooorjem in na navzdol obrnjeni strani s 3’-koncem kunčjega beta-globinskega gena, ki vključuje drugi intron z • — 23;—-----njegovimi primernimi signali za vezanje in poliadenilacijsko zaporedje. Nadalje vsebujejo zaporedja, ki kodirajo na neomicin rezistenten gen iz transposona Tn5 ali v danem primeru iz Tn9, ali sekvence, ki kodirajo higromicin fosfotransferazo,priležno obdano na navzgor usmerjeni strani zapored z zgodnjim promotorjem iz SV40 virusa, ki vključuje’ tudi SV40 začetek replikacije, in naravni promotor Tn5 neo gena, ter po navzdol usmerjeni stra ni s segmentom SV40 zgodnjega gena, ki vključuje majhne t-antigenske vezne in poliadenilacijske signale. Celotno konstrukcijo kloniramo v fragmentu plazmida E.coli pBR322, ki vključuje plazmidni začetek podvojevanja, ampicilin rezistenten gen, toda manjkajo mu t.im. zastrupijevalna zaporedja, ki inhibirajo SV40-način DNA podvojevanja v celicah sesalcev. Po izbiri vključimo v vektor gen, ki kodira dihidrofolat reduktazo (DHFR); prednostno se uporablja modularni DHFR gen, opisan v R.J. Kaufman et al. /Mol. Celi. Bio. 2, 1304-1319 (1982)/. Ta modularni DHFR gen sestoji iz zaporedoma še večjega poznega promotorja adenovirusa tipa 2, fragmenta gena za imunoglobulin, CDNA, ki kodira del DHFR glodalcev in SV40 začetno poliadenilacijsko mesto.Preferred hybrid vectors for use in mammalian cells comprise a cDNA of a hybrid PA or mutants of a hybrid PA, operably flanked on the upstream side by the cytomegalovirus murine immediate-early gene enhancer-promoter and on the downstream side by the 3' end of the rabbitbeta -globin gene, which includes the second intron with its appropriate splice and polyadenylation sequence signals. They further contain sequences coding for the neomycin resistance gene from the Tn5 transposon or, optionally, from Tn9, or sequences coding for hygromycin phosphotransferase, flanked on the upstream side by the early promoter from the SV40 virus, which also includes the SV40 origin of replication, and the natural promoter of the Tn5 neo gene, and on the downstream side by a segment of the SV40 early gene, which includes the small t-antigen binding and polyadenylation signals. The entire construct is cloned into a fragment of the E. coli plasmid pBR322, which includes the plasmid origin of replication, the ampicillin resistance gene, but lacks the so-called poisoning sequences that inhibit the SV40 mode of DNA replication in mammalian cells. Optionally, a gene coding for dihydrofolate reductase (DHFR) is included in the vector; preferably, the modular DHFR gene described in RJ Kaufman et al. /Mol. Cell. Bio. 2, 1304-1319 (1982). This modular DHFR gene consists of a sequentially larger adenovirus type 2 late promoter, an immunoglobulin gene fragment, a cDNA encoding a portion of rodent DHFR, and an SV40 polyadenylation initiation site.
Novi prednostni hibridni vektorji za uporabo v celicah sesalcev predstavljajo napredek v stroki. So boljši v primerjavi z doslej znanimi vektorji v tem, da vsebujejo močan ekspresijski signal za klonirano cDNA, ki se nahaja v mišjem citomegalovirusu nenadno-zgodnjem promotorju/pospeševalniku in v beta-globin zaporedjih za vezanje/poliadenilacijo v okolju, ki dovoljuje ekspresijo na visokem nivoju v ekstremno široki množici celičnih vrst vretenčarjev. Bolj specifično se vektor24 ji lahko uporabljajo (a) da se izrazi cDNA začasno v normalnih t.j. ne SV40-transformiranih celičnih linijah tkivne kulture, toda (b) celo bolje z večjim številom kopij v celicah primatov, ki izražajo SV40T-antigen, tako, da dovoljujejo vektorju,da se podvojuje preko svojega SV40 začetka podvojevanja, ampak tudi (c) da se izrazi taka klonirana cDNA stabilno v celičnih linijah normalne tkivne kulture, kjer se vektor lahko integrira v kromosom celice gostitelja in (d) celo bolje, zaradi večjega števila kopij, če vpeljemo vektor v SV40T-antigen, ki ga proizvajajo celične linije primatov, kjer se vektor lahko podvojuje episomalno.The novel preferred hybrid vectors for use in mammalian cells represent an advance in the art. They are superior to previously known vectors in that they contain a strong expression signal for the cloned cDNA located within the murine cytomegalovirus immediate-early promoter/enhancer and beta-globin binding/polyadenylation sequences in an environment that permits high-level expression in an extremely broad range of vertebrate cell types. More specifically, the vector24 can be used (a) to express cDNA transiently in normal, i.e. not in SV40-transformed tissue culture cell lines, but (b) even better at higher copy number in primate cells expressing SV40T-antigen, by allowing the vector to replicate via its SV40 origin of replication, but also (c) to express such cloned cDNA stably in normal tissue culture cell lines, where the vector can integrate into the host cell chromosome, and (d) even better, due to higher copy number, if the vector is introduced into SV40T-antigen producing primate cell lines, where the vector can replicate episomally.
Povečevalno-promotorski del MCMV obsega npr. DNA z začetkom pri nukleotidih -835 do -443 in konec pri nukleotidu +50 (šteto od mRNA začetka) na 5’ delu MCMV večjega nenadnega zgodnjega gena. Prednostni pospeševalni promotorski del MCMV obsega nukleotide -542 do +50.The MCMV enhancer-promoter portion comprises, for example, DNA starting at nucleotides -835 to -443 and ending at nucleotide +50 (counting from mRNA start) on the 5' portion of the MCMV major immediate early gene. A preferred MCMV enhancer-promoter portion comprises nucleotides -542 to +50.
3’ priležni del gena za ^-globin zajca sestoji iz druge polovice gena za beta-globin zajca /P. Dierks et al., Proč. Natl. Acad. Sci. USA 78, 1411-1415 (1981); A. van Ooyen et al., Science 206, 337-344 (1979)/, začne v drugem eksonu, prednostno na BamHI mesta, tako vključuje drugi intron s signali za veza vo na svojih priležnih zaporedjih, in se zaključi z- poliadenilacij skimi signali, prednostno na Bglll mestu, ki se nahaja 1.2 kb za zgornjim BamHI mestom.The 3' flanking portion of the rabbit β-globin gene consists of the second half of the rabbit beta-globin gene /P. Dierks et al., Proc. Natl. Acad. Sci. USA 78, 1411-1415 (1981); A. van Ooyen et al., Science 206, 337-344 (1979)/, begins in the second exon, preferably at the BamHI site, thus including the second intron with splicing signals on its flanking sequences, and ends with polyadenylation signals, preferably at the BglII site, located 1.2 kb downstream of the upstream BamHI site.
SV40 izvor podvojevanja se nahaja npr. v Hindlll-SphI fragmentu virusne DNA /nukleotidi 5171 do 128, začetek = lega 1; Tooze J. (ed.) DNA Tumor Viruses, Part. 2, 2nd Edition,The SV40 origin of replication is located, for example, in the HindIII-SphI fragment of viral DNA /nucleotides 5171 to 128, start = position 1; Tooze J. (ed.) DNA Tumor Viruses, Part. 2, 2nd Edition,
- 25 Cold Spring Harbor Laboratory, Cold Spring Harbor Ν.Υ. 1982/. Prednostna izvedba je kakorkoli Hindlll-Hpall fragment (nukleotidi 5171 do 346), ki poleg začetka podvojevanja vsebuje še virusni pospeševalnik/promotor, uporaben za pospeševanje prepisovanja selekcijskega gena vektorja.- 25 Cold Spring Harbor Laboratory, Cold Spring Harbor NY. 1982/. A preferred embodiment is any HindIII-Hpall fragment (nucleotides 5171 to 346) which, in addition to the origin of replication, also contains a viral enhancer/promoter useful for promoting transcription of the vector selection gene.
Neomicinski gen se klonira za promotorjem, ki je aktiven v celicah tkivne kulture, prednostno SV40 zgodnji promotor, npr. nameščen na Hpall-Hindlll fragmentu,omenjeno zgoraj. Kodirna zaporedja neomicinskega gena so vsebovana npr. na Bglll Smal fragmentu iz transpozona Tn5 /E. Beck et al., Gene 19, 327-336 (1982); P. Southern et al., J. Mol. Appl. Genet. 1, 327-341 (1982); F. Colbere-Garapin et al., J. Mol. Biol. J50,The neomycin gene is cloned behind a promoter that is active in tissue culture cells, preferably the SV40 early promoter, e.g. located on the Hpall-HindIII fragment, mentioned above. The coding sequences of the neomycin gene are contained e.g. on the Bglll SmaI fragment from the Tn5 transposon /E. Beck et al., Gene 19, 327-336 (1982); P. Southern et al., J. Mol. Appl. Genet. 1, 327-341 (1982); F. Colbere-Garapin et al., J. Mol. Biol. J50,
1-14 (1981)/. Prednostno je, da opremimo neomicinski gen z drugim promotorjem, ki tudi dovoljuje prepisovanje v E.coli. Npr., naravni promotor Tn5 neomicinskega gena, ki je prednostno vsebovan na Hindlll-Bglll fragmentu, lahko namestimo za evkario.ntskim promotorjem pred neo kodirna zaporedja (Southern et al., zgoraj) ali nadalje navzgor pred evkariontski promotor (Colbere Garapin et al., zgoraj). Da se izrazi v celicah tkivne kulture, mora bakterijskemu neo genu slediti poliadenilacijski signal, prednostno del SV40t antigenskega gena, ki tudi vsebuje vezavne signale. Zaporedje, ki kodira neomicin fosfotransferazo, zla sti BgHI-Smal del Tn5 fragmenta, omenjenega zgoraj, lahko tudi nadomestimo z zaporedjem, ki kodira higromicin B fosfotransferazo, prednostno v obliki BamHI fragmenta plazmida pLG89 /L. Gritz et al., Gene 25, 179-188 (3983)/, ki ga lahko bolj primer no vključimo v pSVd /Luedin et al., EMBO-J. 6, 109-114 (1987), derivat iz pSV2911 neo, v katerem je vpeljan Bglll povezovalec na Smal mestu v vektorju.1-14 (1981)/. It is preferable to equip the neomycin gene with another promoter which also permits transcription in E. coli. For example, the native Tn5 promoter of the neomycin gene, which is preferably contained on a HindIII-BglII fragment, can be placed after the eukaryotic promoter before the neo coding sequences (Southern et al., supra) or further upstream before the eukaryotic promoter (Colbere Garapin et al., supra). In order to be expressed in tissue culture cells, the bacterial neo gene must be followed by a polyadenylation signal, preferably a part of the SV40t antigen gene, which also contains the binding signals. The sequence encoding neomycin phosphotransferase, in particular the BgHI-SmaI part of the Tn5 fragment mentioned above, can also be replaced by a sequence encoding hygromycin B phosphotransferase, preferably in the form of a BamHI fragment of plasmid pLG89 /L. Gritz et al., Gene 25, 179-188 (3983)/, which can be more conveniently incorporated into pSVd /Luedin et al., EMBO-J. 6, 109-114 (1987), a derivative of pSV2911 neo in which a BglII linker is introduced at the SmaI site in the vector.
Naslednji prednostni selekcijski gen vključuje zaporedje za kodiranje encima dihidrofolat reduktaze, kot v pSV2dhfr ATCC 37145), ki ne dopušča samo selekcije transformiranih celičnih vrst,ampak tudi pojačanje s plazmidom združenega DNA zaporedja, pogosto s proporcionalnim povečanjem produkcije proteinov v smislu izuma, kodiranih v plazmidu.Another preferred selection gene includes a sequence encoding the enzyme dihydrofolate reductase, as in pSV2dhfr ATCC 37145), which allows not only the selection of transformed cell lines, but also the amplification of the plasmid-associated DNA sequence, often with a proportional increase in the production of the proteins of the invention encoded in the plasmid.
Fragment, ki izhaja iz plazmida pBR322 E.coli, vključuje pBR322 začetek podvojevanja in ampicilin rezistentni gen. Fragment je prednostno vzet iz pBR322 derivata, kot je pSVOd /P. Mellon et al., Celi 27, 279-288 (1981)/, v katerem je odstranjeno t.im. zastrupljevalko zaporedje, ki bi inhibiralo SV40T antigen-vodeno podvojevanje vektorja.The fragment derived from the plasmid pBR322 E. coli includes the pBR322 origin of replication and the ampicillin resistance gene. The fragment is preferably taken from a pBR322 derivative, such as pSVOd /P. Mellon et al., Cell 27, 279-288 (1981)/, in which the so-called toxic sequence that would inhibit SV40T antigen-directed replication of the vector has been removed.
V prednostni izvedbi se predloženi izum nanaša na hibridne vektorje, ki so zmožni podvojevanja in fenotipične selekcije v evkariontskem gostiteljskem soju, ki obsegajo promotor in DNA, ki kodirajo hibridni PA ali mutante hibridnega PA; imenovane DNA se nahajajo skupaj s signali za prepisovanje in terminacijo kot tudi A signali za začetek in konec translacije v imenovanem hibridnem vektorju pod kontrolo imenovanega promotorja; kot oni v transformiranem gostitelju se izraža v proizvajanju proteina.In a preferred embodiment, the present invention relates to hybrid vectors capable of replication and phenotypic selection in a eukaryotic host strain, comprising a promoter and DNA encoding a hybrid PA or mutants of a hybrid PA; said DNAs are located together with transcription and termination signals as well as A signals for the initiation and termination of translation in said hybrid vector under the control of said promoter; as such, in a transformed host, it is expressed to produce a protein.
Hibridne vektorje v smislu izuma pripravljamo po metodah, znanih v stroki, npr. z vezavo DNA segmentov, ki vsebujejo promotor, območjem za kodiranje hibridnega PA ali mutante hidridnega PA, 3’ priležnim zaporedjem in vektorsko DNA.Hybrid vectors according to the invention are prepared by methods known in the art, e.g. by ligation of DNA segments containing a promoter, a region encoding a hybrid PA or a mutant of a hybrid PA, a 3' flanking sequence and vector DNA.
Za vezavo DNA segmentov in vitro uporabljamo lahko različne tehnike. Tope konce (popolnoma komplementarni DNA dupleks), ki jih tvori jo določene restrikcijske endonukleaze, lahko neposredno povežemo s T4 DNA ligazo. Bolj običajno, DNA segmente poveže mo preko njihovih enovlaknatih kohezivnih koncev in kovalentno zapremo z DNA ligazo npr. T4DNA ligazo. Taki enovlaknati kohezivni konci lahko nastanejo pri cepljenju DNA z drugo vrsto endonukleaz, ki proizvajajo odprte konce (dva vlakna DNA dupleksa se cepita na različnih točkah v razdalji nekaj nukleotidov). Enojna vlakna lahko nastanejo tudi z dodajanjem nukleotidov k topim koncem ali odprtim koncem z uporabo terminalnih transferaz (homopolimerni repi) ali z enostavno digestijo enega vlakna na DNA segmentu s topim koncem s primerno eksonukleazo, kotz\ — eksonukleazo. Nadaljni pristop k tvorbi odprtih koncev sestoji iz povezave segmentov DNA s topimi konci s kemično sintetizirano povezalno DNA, ki vsebuje spoznavno mesto za endonukleazo, ki tvori odprti konec,in digestijo rezultirajoče DNA z odgovarjajočo endonukleazo. Komponente hibridnih vektorjev v smislu izuma povežemo skupaj v prej določenem redu, da zagotovimo pravilno delovanje.Various techniques can be used to join DNA segments in vitro. Blunt ends (fully complementary DNA duplexes) formed by certain restriction endonucleases can be directly ligated with T4 DNA ligase. More commonly, DNA segments are joined via their single-stranded cohesive ends and covalently sealed with a DNA ligase such as T4DNA ligase. Such single-stranded cohesive ends can be formed by cleavage of DNA with another type of endonuclease that produces open ends (the two strands of a DNA duplex are cleaved at different points a few nucleotides apart). Single strands can also be formed by adding nucleotides to blunt ends or open ends using terminal transferases (homopolymer tails) or by simply digesting one strand of a blunt-ended DNA segment with a suitable exonuclease, suchas \ — exonuclease. A further approach to the formation of open ends consists of linking blunt-ended DNA segments with chemically synthesized linker DNA containing a recognition site for the open-end-forming endonuclease, and digesting the resulting DNA with the appropriate endonuclease. The components of the hybrid vectors of the invention are ligated together in a predetermined order to ensure proper function.
Gostitelji, transformirani s hibridnimi vektorji, ki vsebujejoHosts transformed with hybrid vectors containing
DNA za hibridni PADNA for hybrid PA
Nadaljni vidik predloženega izuma vključuje evkariontske gostiteljske organizme, transformirane s hibridnimi vektorji, ki obsegajo DNA, ki kodira hibridni PA, ki je sestavljen iz najmanj dveh podzaporedij, ki ustrezajo po identiteti in številu amino kislin podzaporedjem humanega u-PA in humanega t-PA ali ki kodirajo njegovo mutanto in mutante imenovanega gostitelja, in na postopke za njihovo pripravo.A further aspect of the present invention includes eukaryotic host organisms transformed with hybrid vectors comprising DNA encoding a hybrid PA consisting of at least two subsequences corresponding in identity and amino acid number to the subsequences of human u-PA and human t-PA or encoding a mutant thereof and mutants of said host, and methods for their preparation.
Primeri ustreznih evkariontskih gostiteljev so tisti, ki so navedeni zgoraj, zlasti soji kvasa in celice sesalcev. Mutante transformiranih gostiteljskih organizmov vključujejo zlasti mutante, ki imajo malo .proteaz, ki razgrajujejo hibride PA ali mutante hibridnega PA ter dajo višji donos hibridnega PA oz. mutant hibridnega PA.Examples of suitable eukaryotic hosts are those listed above, in particular yeast strains and mammalian cells. Mutants of transformed host organisms include in particular mutants that have low levels of proteases that degrade hybrid PA or hybrid PA mutants and give a higher yield of hybrid PA or hybrid PA mutant.
Postopek za pripravo transformiranih evkariontskih gostiteljev obsega transformiranje ali transfekcijo. evkariontskega gostitelja z ekspresijskim vektorjem, ki obsega DNA izuma, regulirano z ekspresijskim kontrolnim zaporedjem.The method for preparing transformed eukaryotic hosts comprises transforming or transfecting a eukaryotic host with an expression vector comprising DNA of the invention regulated by an expression control sequence.
Transformacija evkariontskih gostiteljskih celic je izvedena z metodami, znanimi v stroki. Npr. transformacija kvasa s hibridnimi vektorji se lahko izvede v smislu metode, ki jo je opisal Hinnen in sodelavci /Proč. Natl. Acad. Sci. USA 75,Transformation of eukaryotic host cells is carried out by methods known in the art. For example, transformation of yeast with hybrid vectors can be carried out in accordance with the method described by Hinnen et al. /Proc. Natl. Acad. Sci. USA 75,
1919 (1978)/. Ta metoda se lahko razdeli v tri stopnje:1919 (1978)/. This method can be divided into three stages:
1) Odstranitev celične stene kvasovk ali njenih delov.1) Removal of the yeast cell wall or parts thereof.
2) Obdelava golih kvasovk (sferoplastov) s transformirajočo i2) Treatment of naked yeast (spheroplasts) with transforming i
DNA v prisotnosti PEG (polietilenglikol) in Ca^ ionov.DNA in the presence of PEG (polyethylene glycol) and Ca^ ions.
3) Regeneracija celične stene in selekcija transformiranih celic v trdni sloj agarja.3) Cell wall regeneration and selection of transformed cells into a solid agar layer.
Prednostne metode:Preferred methods:
ad (1): Celično steno kvasovk odstranimo encimatsko z uporabo različnih pripravkov glukozidaz, kot so sokovi črevesa polža (npr. Glusulase®ali Helicase^ ali encimskih mešanic, pridobljenih iz mikroorganizmov (npr. Zvmolvase^ v osmoznoad (1): The yeast cell wall is removed enzymatically using various preparations of glucosidases, such as snail intestinal juices (e.g. Glusulase® or Helicase^ or enzyme mixtures obtained from microorganisms (e.g. Zvmolvase^ in osmotic
- 29stabiliziranih raztopinah (npr. 1 M sorbitol).- 29stabilized solutions (e.g. 1 M sorbitol).
ad (2): Sferoplasti kvasa se kopičijo v prisotnosti PEG, kar privede do lokalnih fuzij- zlitij citoplazemskih membran. Nastajanje zlitju podobnih pogojev je odločilno in mnogo transformiranih celic kvasa postane diploidnih ali celo triploidnih med procesom transformacije. Postopki, ki dovoljujejo selekcijo zlitih sferoplastov, se lahko uporabljajo za obogatitev transformantov,npr. transformirane celice se lahko brez težav ločijo med preselekcioniranimi produkti zlitja.ad (2): Yeast spheroplasts accumulate in the presence of PEG, leading to local fusions of the cytoplasmic membranes. The formation of fusion-like conditions is crucial and many transformed yeast cells become diploid or even triploid during the transformation process. Procedures that allow the selection of fused spheroplasts can be used to enrich transformants, e.g. transformed cells can be easily separated from preselected fusion products.
ad (3): Ker se kvasovke brez celične stene ne delijo, se mora celična stena obnoviti. To obnovo primerno izvedemo z vklapljanjem sferoplastov v agar. Npr. staljeni agar (okrog 50°C) pomešamo s sferoplasti. Pri ohlajanju raztopine do temperature rasti kvasa (okoli 30°C) dobimo trden sloj. Ta sloj agarja preprečuje hitro difuzijo·. in izgubo bistvenih makromolekul iz sferoplastov in zato pospešuje obnovo celične stene. Vendar pa lahko obnovo celične stene dosežemo tudi (čeprav z manjšim izkoristkom) z nasaditvijo sferoplastov na površino predhodno formiranih plasti agarja.ad (3): Since yeasts without a cell wall do not divide, the cell wall must be renewed. This renewal is conveniently carried out by incorporating spheroplasts into agar. For example, molten agar (around 50°C) is mixed with spheroplasts. When the solution is cooled to the yeast growth temperature (around 30°C), a solid layer is obtained. This agar layer prevents rapid diffusion and loss of essential macromolecules from the spheroplasts and therefore accelerates the renewal of the cell wall. However, cell wall renewal can also be achieved (although with lower efficiency) by seeding spheroplasts onto the surface of previously formed agar layers.
Prednostno pripravimo regeneracijski agar na način, ki dovoljuje istočasno obnovo in selekcijo transformiranih celic. Ker se geni kvasa, ki kodirajo encime amino kislinskih biosinteznih poti, v glavnem uporabljajo kot selektivni označevalci (supra), regeneracijo prednostno izvedemo v minimalnem agarskem mediju za kvas. Če je zahteva po zelo visokih učinkih regeneracije, je koristen naslednji dvostopenjski postopek: (1) regeneracija celične stene v bogatem kompleksnem mediju in (2) selekcija transformiranih celic z replično nasaditvijo celičnega sloja v selektivna gojišča iz agarja.Preferably, the regeneration agar is prepared in a manner that allows simultaneous regeneration and selection of transformed cells. Since yeast genes encoding enzymes of the amino acid biosynthetic pathways are mainly used as selectable markers (supra), regeneration is preferably carried out in yeast minimal agar medium. If very high regeneration efficiencies are required, the following two-step procedure is useful: (1) cell wall regeneration in a rich complex medium and (2) selection of transformed cells by replica plating of the cell layer onto selective agar media.
Vpeljava hibridnih vektorjev v celice sesalcev je izvedena s transfekcijo v prisotnosti pomožnih spojin, npr. dietilaminoetildekstrana, dimetil sulfoksida, glicerola, polietilenglikola ali podobnim ali kakor ko-precipitatorji vektorske DNA, in kalcijevega fosfata. Nadaljnje primerne metode vključujejo neposredno mikroinjiciranje vektorske DNA v celično jedro in z elektrovnašanjem, npr. uvedbo DNA s kratkimi električnimi pulzi, ki povečujejo permeabilnost celičnih membran. Sledečo selekcijo transformiranih celic lahko izvedemo z uporabo selekcijskega označevalca, ki je ali kovalentno integriran v ekspresijski vektor, ali dodan kot ločena enota. Selekcijski označevalci vključujejo gene, ki podelijo odpornost do antibiotikov,ali gene, ki popravijo genetično poškodbo gostiteljske celice (supra). Prednosten selekcijski sistem uporablja celice, ki jim manjka dihidrofolat reduktaza (DHRF-), npr. CHO celice, ki absolutno zahtevajo timidin, glicin in purine za rast, razen, če je dodan eksogeni DHFR gen. Ob uvedbi vektorja, ki vsebuje gen hibridnega PA in dodatno DHFR gen, v ustrezne DHFR- celice, npr. CHO celice,, transformirane celice ločimo s povečanjem koncentracije antifolatnega zdravila metothrexata v mediju.The introduction of hybrid vectors into mammalian cells is carried out by transfection in the presence of adjuvants, e.g. diethylaminoethyldextran, dimethyl sulfoxide, glycerol, polyethylene glycol or the like or as co-precipitants of the vector DNA, and calcium phosphate. Further suitable methods include direct microinjection of the vector DNA into the cell nucleus and by electroporation, e.g. introduction of the DNA by short electrical pulses which increase the permeability of the cell membranes. Subsequent selection of transformed cells can be carried out using a selectable marker, which is either covalently integrated into the expression vector or added as a separate unit. Selectable markers include genes which confer resistance to antibiotics, or genes which repair genetic damage to the host cell (supra). A preferred selection system uses cells which lack dihydrofolate reductase (DHRF- ), e.g. CHO cells, which absolutely require thymidine, glycine and purines for growth unless an exogenous DHFR gene is added. Upon introduction of a vector containing the hybrid PA gene and an additional DHFR gene into appropriate DHFR- cells, e.g. CHO cells, the transformed cells are separated by increasing the concentration of the antifolate drug methotrexate in the medium.
Posebno prednostna je selekcijska metoda, pri kateri ustrezne celice sesalcev, npr. CHO celice,obdelamo s ko-precipitati vektorske DNA, ki vsebuje gen hibridnega PA,in gen, ki kodira antibiotično odpornost, npr. odpornost do G—418 in kalci31 jev fosfat. Transformirane celice ločimo z gojenjem v prisotnosti odgovarjajočih antibiotikov, npr. G-418 in/ali s skriningom za ekspresijo hibridnega PA.,A particularly preferred selection method is one in which suitable mammalian cells, e.g. CHO cells, are treated with co-precipitates of vector DNA containing the hybrid PA gene and a gene encoding antibiotic resistance, e.g. resistance to G-418 and calcium phosphate. Transformed cells are separated by culturing in the presence of appropriate antibiotics, e.g. G-418 and/or by screening for expression of the hybrid PA.
Transformirane gostiteljske organizme v smislu izuma lahko izboljšamo v izdelovanju hibridnih PA ali mutant hibridnih PA z uporabnimi metodami mutacije in selekcije, znanimi v stroki. Mutacijo lahko izvedemo npr. z UV-obsevanjem ali primernimi kemičnimi substancami. Zlasti prednostno je izdelovanje proteazno-pomanjkljivih mutant, zlasti mutant kvasa, zato, da odpravimo proteolitično razgradnjo proizvedenega hibridnega PA oz. mutante hibridnega PA.Transformed host organisms according to the invention can be improved in the production of hybrid PA or mutant hybrid PA by useful mutation and selection methods known in the art. Mutation can be carried out e.g. by UV irradiation or suitable chemical substances. It is particularly preferred to produce protease-deficient mutants, especially yeast mutants, in order to eliminate proteolytic degradation of the produced hybrid PA or mutant hybrid PA.
Gojenje transformiranih gostiteljskih celicCultivation of transformed host cells
Nadalje se izum nanaša na metodo za pridobivanje enoverižnih hibridnih PA, ki imajo amino kislinsko zaporedje, sestavljeno iz najmanj dveh podzaporedij, ki ustrezata po identiteti in številu amino kislin podzaponedjema humanega t-PA in humanega u-PA ali njihovim mutantom, ki vključuje gojenje transformiranega evkariontskega gostitelja pod primernimi hranilnimi pogoji, ki vsebuje DNA zaporedja, ki kodira imenovani hibridni PA ali mutanto hibridnega PA,in na izoliranje imenovanega hibridnega PA ali njegove mutante. Transformirane gostiteljske celice gojimo po metodah, znanih v stroki, v tekočem mediju, ki vsebuje vire ogljika in dušika, ki se lahko asimilirajo ten anorganske soli.The invention further relates to a method for obtaining single-chain hybrid PAs having an amino acid sequence consisting of at least two subsequences corresponding in identity and number of amino acids to the subsequences of human t-PA and human u-PA or mutants thereof, which comprises culturing a transformed eukaryotic host under suitable nutrient conditions containing a DNA sequence encoding said hybrid PA or a mutant of said hybrid PA, and isolating said hybrid PA or its mutants. The transformed host cells are cultured according to methods known in the art in a liquid medium containing carbon and nitrogen sources that can be assimilated from inorganic salts.
Za gojenje transformiranih kvasovk v smislu izuma se lahko uporabljajo različni -viri ogljika. Primeri prednostnih virov za ogljik so ogljikovi hidrati, ki se lahko asimilirajo, kot so glukoza, maltoza, manitol ali laktoza ali acetat, kateri se lahko uporaljat ali sam ali v primernih mešanicah. Primeri ustreznih virov dušika so amino kisline kot so kezamino kisline, peptidi in proteini ter njihovi razgradni produkti, kot so tripton, pepton ali mesni izvlečki, izvlečki kvasa, izvleček slada in tudi amonijeve soli, npr. amonijev klorid, sulfat ali nitrat, ki se lahko uporabajo kot taki ali v primernih mešanicah. Anorganske soli, ki se lahko tudi uporabljajo, so npr. sulfati, kloridi, fosfati in karbonati natrija, kalija, magnezija in kalcija. Medij nadalje vsebuje npr. snovi, ki pospešujejo rast, kot so sledovi elementov, npr. železo, cink, mangan ipd., in prednos tno substance, ki uveljavljajo selekcijski pritisk in preprečujejo rast celic, ki so izgubile ekspresijo plazmida. Tako npr., če se uporablja kot gostiteljski mikroorganizem soj kvasovk, ki je avksotrofen v npr. esencialni amino kislini, vsebuje plazmid prednostno gen za kodiranje encima, ki dopolni pomanjkljivost gostitelja. Gojenje vrste kvasovk poteka v minimalnem mediju, nepopolnem v imenovani amino kislini.Various carbon sources can be used for the cultivation of the transformed yeasts according to the invention. Examples of preferred carbon sources are assimilable carbohydrates, such as glucose, maltose, mannitol or lactose or acetate, which can be used either alone or in suitable mixtures. Examples of suitable nitrogen sources are amino acids such as ketamine acids, peptides and proteins and their degradation products, such as tryptone, peptone or meat extracts, yeast extracts, malt extract and also ammonium salts, e.g. ammonium chloride, sulphate or nitrate, which can be used as such or in suitable mixtures. Inorganic salts which can also be used are e.g. sulphates, chlorides, phosphates and carbonates of sodium, potassium, magnesium and calcium. The medium further contains e.g. substances which promote growth, such as trace elements, e.g. iron, zinc, manganese, etc., and preferably substances that exert selection pressure and prevent the growth of cells that have lost plasmid expression. For example, if a yeast strain is used as a host microorganism that is auxotrophic in, for example, an essential amino acid, the plasmid preferably contains a gene encoding an enzyme that supplements the host deficiency. The yeast strain is grown in a minimal medium deficient in the said amino acid.
Gojenje izvajamo po postopkih, ki so znani v stroki.Cultivation is carried out according to procedures known in the art.
Pogoji gojenja, kot so temperatura, pH vrednost medija in čas fermentacije.so izbrani tako, da dobimo maksimalen titer PA proteinov izuma. Tako soj kvasovk prednostno gojimo pod aerobnimi pogoji s potopitveno kulturo ob stresanju ali mešanju pri temperaturi okoli 20 do 40°C, prednostno okoli 30°C in pH vrednostjo od 5 do 8, prednostno okoli pH 7 za okoli 4 do 30 ur, prednostno dokler ne dosežemo maksimalnega donosa proteinov izuma.The cultivation conditions, such as temperature, pH of the medium and fermentation time, are selected to obtain a maximum titer of the PA proteins of the invention. Thus, the yeast strain is preferably cultivated under aerobic conditions by submerged culture with shaking or stirring at a temperature of about 20 to 40°C, preferably about 30°C and a pH of from 5 to 8, preferably about pH 7 for about 4 to 30 hours, preferably until a maximum yield of the proteins of the invention is achieved.
- 33 Celice sesalcev rastejo pod pogoji tkivne kulture v medijih, ki so komercialno na razpolago, po izbiri dopolnjeni s snovmi, ki pospešujejo rast in/ali serumi sesalcev. Celice rastejo ali pritrjene na trdno podlago npr. mikronosilcih ali poroznih steklenih vlaknih, ali prosto plavajoče v ustreznih gojitvenih posodah. Gojitveni medij izberemo na tak način, da je uveljavljen selekcijski pritisk in da preživijo le tiste celice, ki še vsebujejo hibridni vektor DNA, ki vključuje genetični marker. Tako npr. dodamo v medij antibiotik, če hibridni vektor vključuje odgovarjajoči gen za antibiotično odpornost. Ko je dosegla gostota celic zadovoljivo vrednost, gojenje prekinemo in protein izoliramo. Če uporabljamo celice sesalcev se hibridni PA ali mutanta hibridnega PA običajno izloča v medij. Medij,ki vsebuje produkt, ločimo od celic, katere - oskrbljene s svežim medijem — uporabljamo za kontinuirno proizvodnjo. Če uporablja mo kvasovke, se lahko protein akumulira tudi znotraj celic, posebno v periplazmatskem prostoru. V tem primeru prva stopnja za pridobivanje PA proteina sestoji iz sproščanja proteina iz celične notranjosti. Pri večini postopkov se najprej odstrani celična stena z encimatsko razgradnjo celične stene z glukozidazami (supra). Alternativno se celična stena odstrani z obdelavo s kemičnimi sredstvi, npr. tiolnimi reagenti ali EDTA, ki povzročijo poškodbe celične stene, kar omogoča, da se proizveden hibridni PA ali njegova mutanta sprosti. Rezultirajočo mešanico obogatimo na hibridnem PA ali njegovi mutanti po običajnem načinu, kot je odstranitev večine neproteinskega materiala z obdelavo s polietileniminom, preeipitacijo proteinov z amonijevim sul- 34 fatom, gel elektroforezo, dializo, kromatografijo, npr. kromatografijo na osnovi ionske izmenjave, kromatografijo na osnovi ločevanja po velikosti, HPLC ali HPLC z reverzno fazo, molekularnim sortiranjem na ustrezni Sephadex koloni ali podobno. Končno čiščenje predhodno očiščenega produkta dosežemo npr. z afinitetno kromatografijo, npr. afinitetno kromatografijo na principu protiteles, zlasti afinitetno kromatografijo na principu monoklonalnih protiteles, ki uporablja monoklonalna anti-tPA ali anti-u-PA protitelesa, pritrjena na netopen matriks, po metodah, znanih v stroki^ali v primeru hibridnih PA,ki vsebujejo katalitično B-verigo t-PA, DE-3 afinitetno kromatografijo (DE-3 je proteazni inhibitor, izoliran iz Erytrina latissima) in podobno. Predmet izuma so tudi hibridne celične linije, ki proizvajajo monoklonalna protitelesa, usmerjena na specifične domene t-PA ali u-PA,in imenovana monoklonalna protitelesa.- 33 Mammalian cells are grown under tissue culture conditions in commercially available media, optionally supplemented with growth promoting substances and/or mammalian serum. The cells are grown either attached to a solid support, e.g. microcarriers or porous glass fibers, or free-floating in suitable culture vessels. The culture medium is chosen in such a way that selection pressure is exerted and only those cells that still contain the hybrid DNA vector that includes the genetic marker survive. For example, an antibiotic is added to the medium if the hybrid vector includes the corresponding antibiotic resistance gene. When the cell density has reached a satisfactory value, the culture is stopped and the protein is isolated. If mammalian cells are used, the hybrid PA or a mutant of the hybrid PA is usually secreted into the medium. The medium containing the product is separated from the cells, which - supplied with fresh medium - are used for continuous production. If yeast is used, the protein can also accumulate inside the cells, especially in the periplasmic space. In this case, the first step for obtaining the PA protein consists of releasing the protein from the cell interior. In most processes, the cell wall is first removed by enzymatic degradation of the cell wall with glucosidases (supra). Alternatively, the cell wall is removed by treatment with chemical agents, e.g. thiol reagents or EDTA, which cause damage to the cell wall, allowing the produced hybrid PA or its mutant to be released. The resulting mixture is enriched for the hybrid PA or its mutant by conventional means, such as removal of most non-proteinaceous material by treatment with polyethyleneimine, precipitation of proteins with ammonium sulfate, gel electrophoresis, dialysis, chromatography, e.g. ion exchange chromatography, size exclusion chromatography, HPLC or reverse phase HPLC, molecular sorting on a suitable Sephadex column or the like. Final purification of the previously purified product is achieved e.g. by affinity chromatography, e.g. antibody-based affinity chromatography, in particular monoclonal antibody-based affinity chromatography, which uses monoclonal anti-tPA or anti-u-PA antibodies attached to an insoluble matrix, according to methods known in the art, or in the case of hybrid PAs containing the catalytic B-chain of t-PA, DE-3 affinity chromatography (DE-3 is a protease inhibitor isolated from Erythrina latissima), and the like. The invention also relates to hybrid cell lines that produce monoclonal antibodies directed against specific domains of t-PA or u-PA, and are referred to as monoclonal antibodies.
V ustrezno pripravo eno-verižnega hibridnega PA ali mutante hibridnega PA, ki je bistveno brez dvo-verižne oblike, je koristno vključen proteazni inhibitor, kot je aprotinin Trasylol®) ali bazični tripsin inhibitor iz pankreasa, med postopkom čiščenja zato, da se inhibirajo sledovi proteaz, ki so lahko prisotne v gojilnem mediju in ki lahko povzročijo (delno) pretvorbo eno-verižne oblike v dvo-verižno obliko. Končno čiščenje nato dosežemo s kolonsko kromatografijo, ki vsebuje reagent s selektivno afiniteto.In a suitable preparation of a single-chain hybrid PA or a mutant hybrid PA that is substantially free of the double-chain form, a protease inhibitor, such as aprotinin (Trasylol®) or basic trypsin inhibitor from pancreas, is advantageously included during the purification process in order to inhibit trace proteases that may be present in the culture medium and that may cause (partial) conversion of the single-chain form to the double-chain form. Final purification is then achieved by column chromatography containing a selective affinity reagent.
5. Farmacevtski sestavki5. Pharmaceutical compositions
Novi eno-verižni PA proteini in njihove mutante, ki smo jih dobili v smislu predloženega izuma, kažejo pomembne farmakološke lastnosti. Lahko se uporabljajo analogno, kot znani plazminogeni aktivatorji pri ljudeh za preprečevanje ali zdravljenje tromboz in drugih stanj, kjer je zaželeno tvoriti lokalno fibrinolitično ali proteolitično aktivnost preko mehanizma plazminogene aktivacije, kot npr. arterosklerozi, miokardialnem ali cerebralnem infarktu, venozni trombozi, tromboemboliji, postoperativnih trombozah, tromboflebitisu in diabetičnih vaskulopatijah.The novel single-chain PA proteins and their mutants obtained in accordance with the present invention exhibit significant pharmacological properties. They can be used analogously to known human plasminogen activators for the prevention or treatment of thrombosis and other conditions where it is desirable to produce local fibrinolytic or proteolytic activity via the mechanism of plasminogen activation, such as atherosclerosis, myocardial or cerebral infarction, venous thrombosis, thromboembolism, postoperative thrombosis, thrombophlebitis and diabetic vasculopathies.
Presenetljivo smo ugotovili, da novi hibridni PA proteini in njihove mutante v smislu predloženega izuma združujejo koristne lastnosti naravnega t-PA in u-PA. Tako so novi hibridni PA proteini in njihove mutante fibrinolitično aktivni. Edinstvene lastnosti usmerjene na fibrin, npr. zmožnost aktiviranja plazminogena, prednostno v prisotnosti fibrina, so ohranjene. Nadalje imajo novi proteini podaljšano in vivo stabilnost v primerjavi z avtentičnim t-PA.Surprisingly, we have found that the novel hybrid PA proteins and their mutants according to the present invention combine the beneficial properties of native t-PA and u-PA. Thus, the novel hybrid PA proteins and their mutants are fibrinolytically active. The unique fibrin-targeting properties, e.g. the ability to activate plasminogen, preferentially in the presence of fibrin, are retained. Furthermore, the novel proteins have prolonged in vivo stability compared to authentic t-PA.
Izum se nanaša tudi na farmacevtske sestavke, ki obsegajo terapevtsko učinkovito količino aktivne sestavine (hibridnega PA ali njegove mutante), skupaj z organskimi ali anorganskimi, trdnimi ali tekočimi farmacevtsko sprejemljivimi nosil ci, ki so primerni za parenteralno, t.j. intramuskularno, subkutano ali intraperitonealno dajanje in ki ne vplivajo škodljivo na druge aktivne sestavine.The invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of the active ingredient (hybrid PA or its mutant), together with organic or inorganic, solid or liquid pharmaceutically acceptable carriers which are suitable for parenteral, i.e. intramuscular, subcutaneous or intraperitoneal administration and which do not adversely affect other active ingredients.
- 36 Primerne so infuzijske raztopine, prednostno vodne raztopine ali suspenzije, ki jih je mogoče pripraviti pred uporabo npr. iz liofiliziranih preparatov, ki vsebujejo aktivno sestavino samo ali skupaj z nosilcem, kot je manitol, laktoza, glukoza, albumin ipd. Farmacevtski sestavki so sterilizirani in če je zaželeno ,dopolnjeni , npr. s konzervansi, stabilizatorji, emulgatorji, sredstvi za povečanje topnosti, pufri in/ali solmi za regulacijo osmotskega pritiska. Sterilizacijo lahko dosežemo s sterilno filtracijo skozi filtre z majhnimi porami (0,45 premera ali manjšimi), nakar sestavek lahko liofiliziramo, če je zaželeno. Dodajamo lahko tudi antibiotike,zato, da pomagajo pri ohranitvi sterilnosti.- 36 Suitable are infusion solutions, preferably aqueous solutions or suspensions, which can be prepared before use, e.g. from lyophilized preparations containing the active ingredient alone or together with a carrier such as mannitol, lactose, glucose, albumin, etc. The pharmaceutical compositions are sterilized and, if desired, supplemented, e.g. with preservatives, stabilizers, emulsifiers, solubilizers, buffers and/or salts for regulating osmotic pressure. Sterilization can be achieved by sterile filtration through small-pore filters (0.45 diameter or smaller), after which the composition can be lyophilized, if desired. Antibiotics can also be added to help maintain sterility.
Farmacevtski sestavki v smislu predloženega izuma se izdajajo v oblikah enot doziranja, npr. v ampulah, ki vsebujejo 1 do 2000 mg farmacevtsko sprejemljivega nosilca na enoto doze in okoli 1 do 200 mg, prednostno 5 do 100 mg aktivne substance na enoto doziranja.The pharmaceutical compositions of the present invention are presented in dosage unit forms, e.g. in ampoules containing 1 to 2000 mg of pharmaceutically acceptable carrier per dosage unit and about 1 to 200 mg, preferably 5 to 100 mg, of active substance per dosage unit.
Odvisno od vrste bolezni in starosti ter stanja pacienta, znaša dnevna doza, ki jo dajemo pri zdravljenju pacienta, ki tehta približno 70 kg, v območju 3 do 100 mg, prednostno 5 do 50 mg na 24 ur. V primeru miokardialnega infarkta, je dajana dnevna doza prednostno okoli 30 do 80 mg med 60 in 120 minutami, prednostno v treh alikvotih znotraj okoli 90 minut. Celotna količina hibridnega PA ali mutante hibridnega PA se lahko da tudi kot ena injekcija.Depending on the type of disease and the age and condition of the patient, the daily dose administered in the treatment of a patient weighing approximately 70 kg is in the range of 3 to 100 mg, preferably 5 to 50 mg per 24 hours. In the case of myocardial infarction, the daily dose administered is preferably about 30 to 80 mg over 60 to 120 minutes, preferably in three aliquots over about 90 minutes. The entire amount of hybrid PA or hybrid PA mutant can also be administered as a single injection.
Izum tudi ustvarja metodo za proizvodnjo farmacevtskega sestavka, značilnega v tem, da se biološko aktivni protein v smislu predloženega izuma doda k farmacevtsko sprejemljivem nosilcu .The invention also provides a method for producing a pharmaceutical composition, characterized in that a biologically active protein according to the present invention is added to a pharmaceutically acceptable carrier.
Uporaba novih proteinov za profilaktično in terapevtsko obdelavo človeškega telesa je tudi predmet predloženega izuma.The use of the novel proteins for prophylactic and therapeutic treatment of the human body is also a subject of the present invention.
Izum se nanaša posebej na DNA, hibridne vektorje, transformirane gostiteljske vrste, hibridne PA proteine,mutante hibridnih PA proteinov, hibridne celične linije, monoklonalna protitelesa in na postopke za njihovo pripravo, kot je opisano v primerih.The invention relates particularly to DNA, hybrid vectors, transformed host species, hybrid PA proteins, mutants of hybrid PA proteins, hybrid cell lines, monoclonal antibodies and to processes for their preparation, as described in the examples.
Kratek opis risbBrief description of the drawings
V naslednjem eksperimentalnem delu so opisane razne izvedbe predloženega izuma v zvezi s spremljajočimi risbami, v katerih:In the following experimental part, various embodiments of the present invention are described with reference to the accompanying drawings, in which:
sl. 1 in sl. 3 ponazarjata nukleotidna zaporedja in izvedena zaporedja amino kislin humane t-PA cDNA oz. humane u-PA cDNA. Prve amino kisline zrelih proteinov so podčrtane.Fig. 1 and Fig. 3 illustrate the nucleotide sequences and deduced amino acid sequences of human t-PA cDNA and human u-PA cDNA, respectively. The first amino acids of the mature proteins are underlined.
Sl. 2 in sl. 4 so karte restrikcijskih endonukleaz humane t-PA cDNA oz. humane u-PA cDNA.Fig. 2 and Fig. 4 are restriction endonuclease maps of human t-PA cDNA and human u-PA cDNA, respectively.
Sl. 5 shematsko ponazarja uporabljeno tehniko za konstituiranje plazmida pEcoO.47AScaI.Fig. 5 schematically illustrates the technique used to constitute the plasmid pEcoO.47AScaI.
Sl. 6 shematsko ponazarja konstrukcijo plazmida ph.tPAAScal, ki ga vsebuje mutirana t-PA cDNA.Fig. 6 schematically illustrates the construction of the plasmid ph.tPAAScal, which contains the mutated t-PA cDNA.
Sl. 7 shematsko ponazarja konstrukcijo plazmida pUNC.tc. ki vsebuje vključek cDNA, ki obsega A-verigo domen u-PA in B-verigo t-PA.Fig. 7 schematically illustrates the construction of the plasmid pUNC.tc. containing a cDNA insert comprising the A-chain domains of u-PA and the B-chain of t-PA.
- 38 Sl. 8 shematsko upodablja konstrukcijo plazmida ptNC-UC,ki vsebuje cDNA vključek, ki obsega A-verigo domen t-PA in B-verigo u-PA.- 38 Fig. 8 schematically depicts the construction of the plasmid ptNC-UC, which contains a cDNA insert comprising the A-chain domains of t-PA and the B-chain of u-PA.
Sl. 9 shematsko upodablja konstrukcijo plazmida pD02.Fig. 9 schematically depicts the construction of plasmid pD02.
Sl. 10 shematsko ponazarja konstrukcijo plazmida pD010, ki vsebuje t-PA cDNA, kombinirano s fragmentom //-globina.Fig. 10 schematically illustrates the construction of plasmid pD010 containing t-PA cDNA combined with a β-globin fragment.
Sl. 11 shematsko ponazarja konstrukcijo plazmida pCGA26, ki vsebuje t-PA cDNA pod kontrolo MCMV IE promotorja in fragment β -globina.Fig. 11 schematically illustrates the construction of plasmid pCGA26, which contains t-PA cDNA under the control of the MCMV IE promoter and a β-globin fragment.
Sl. 12 shematsko ponazarja konstrukcijo t-PA ekspresijskega plazmida pCGA28 in splošnega ekspresi jskega plazmida pCGA44·, oba plazmida vključujeta neomicin rezistenten gen.Fig. 12 schematically illustrates the construction of the t-PA expression plasmid pCGA28 and the general expression plasmid pCGA44·, both plasmids including a neomycin resistance gene.
Sl. 13 shematsko ponazarja konstrukcijo t-PA ekspresijskega plazmida pCGA42 in splošnega ekspresijskega plazmida pCGA42d; oba plazmida vključujeta higromicin rezistenten gen.Fig. 13 schematically illustrates the construction of the t-PA expression plasmid pCGA42 and the general expression plasmid pCGA42d; both plasmids include a hygromycin resistance gene.
Sl. 14 shematsko ponazarja konstrukcijo t-PA ekspresijskega plazmida pCGA48, ki vključuje neomicin rezistenten gen in DHFR gen.Fig. 14 schematically illustrates the construction of the t-PA expression plasmid pCGA48, which includes a neomycin resistance gene and a DHFR gene.
Sl. 15 shematsko ponazarja konstrukcijo ekspresijskega plazmida pBR1a, ki vključuje mutiran t-PAcDNA vključek plazmida ph.tPAdScal.Fig. 15 schematically illustrates the construction of the expression plasmid pBR1a, which includes the mutated t-PAcDNA insert of the plasmid ph.tPAdScal.
Sl. 16 shematsko kaže konstrukcijo ekspresijskega plazmida pBR2a, ki vsebuje hibridni PA cDNA vključek, ki obsega A-verigo domen u-PA in B-verigo t-PA.Fig. 16 schematically shows the construction of the expression plasmid pBR2a containing a hybrid PA cDNA insert comprising the A-chain domains of u-PA and the B-chain of t-PA.
Sl. 17 shematsko upodablja konstrukcijo u-PA ekspresijskega plazmida pBR3a.Fig. 17 schematically depicts the construction of the u-PA expression plasmid pBR3a.
pBR4a, ki vsebuje hibridni PA cDNA vključek, ki obsega domene t-PA A-verige in B-verigo u-PA.pBR4a, which contains a hybrid PA cDNA insert comprising the t-PA A-chain and u-PA B-chain domains.
Sl. 19 shematsko kaže konstrukcijo ekspresijskega vektorja kvasa pJDB207/PHQ5-I-TPA, ki vsebuje PHO5 promotor, invertazno signalno zaporedje in t-PA cDNA.Fig. 19 schematically shows the construction of the yeast expression vector pJDB207/PHQ5-I-TPA, which contains the PHO5 promoter, the invertase signal sequence and the t-PA cDNA.
ki obsega u-PA cDNA.comprising u-PA cDNA.
Sl. 22 shematsko kaže konstrukcijo plazmida pJDB207/PHO5-I-UPA.Fig. 22 schematically shows the construction of plasmid pJDB207/PHO5-I-UPA.
Sl. 23-26 shematsko ponazarjajo uporabljene tehnike za pretvorbo konstrukcij primarnih hibridnih PA, ki vključujejo domene A-verige in katalitično območje B-verige u-PA ali t-PA v končno konstrukcijo, v kateri je zveza domen v aktivacijskem mestu in/ali v naravnih veznih mestih ekson-intron.Fig. 23-26 schematically illustrate the techniques used to convert primary hybrid PA constructs that include the A-chain domains and the catalytic region of the B-chain of u-PA or t-PA into a final construct in which the junction of the domains is at the activation site and/or at the natural exon-intron junction sites.
t-PA.t-PA.
- 40 Sl. 26 kaže konstrukcijo gena, ki kodira hibridni PA, ki obsega domeno rastni faktor u-PA, domeno krigle 2 t-PA in B-verigo u-PA.- 40 Fig. 26 shows a gene construct encoding a hybrid PA comprising the u-PA growth factor domain, the t-PA mug 2 domain and the u-PA B-chain.
Sl. 27 je sestavljanje hibridnih PA in mutant hibridnih PA kot so primeri v eksperimentalnem delu.Fig. 27 is the assembly of hybrid PAs and mutant hybrid PAs as examples in the experimental part.
Simboli, uporabljeni v spremljajočih slikah, imajo naslednje pomene:The symbols used in the accompanying figures have the following meanings:
AMP, AmpRTET, TetRNEO TN5PR HPH pBRoriAMP, AmpR TET, TetR NEO TN5PR HPH pBRori
POISSEARCH
SV40oriSV40 ori
SV40enh,SV40E HCMVESV40enh,SV40E HCMVE
MCMVPMCMVP
RSVRSV
CAP polyACAP polyA
SPLDSPLD
SPLASPLA
BAPBAP
CIP (BamHl/Bgl2)CIP (BamH1/Bgl2)
Scal(del) x < y ampicilin rezistenten gen (ft-laktamaza) tetraciklin rezistenten genScal(del) x < y ampicillin resistance gene (ft-lactamase) tetracycline resistance gene
TN5 neomicin fosfotransferaza bakterijski promotor transpozona TN5 higromicin fosfotransferaza začetek -podvojevanja plazmida.pBR322 zastrupijevalno-zaporedje, pBR322 zaporedje, ki inhibira SV40 podvojevanje začetek podvojevanja SV40, ki sovpada z zgodnjimi in poznimi promoto^ji bp povečevalni del SV40 zgodnjega promotorja povečevalnik humanega citomegalovivusnega (HCMV) večjega navadnega zgodnjega gena promotor/mRNA začetno mesto mišjega citomegalovirusnega (MCMC) večjega nenadnega zgodnjega gena Rous sarcoma virus LTR (promotor) lega 5’ m7Gp cap evkariontske mRNA poliadenilacijsko mesto mRNA vezno donorsko mesto, 5’ konec introna vezno akceptorsko mesto, 3’ konec introna bakterijska alkalna fosfataza telečja črevesna fosfatazaTN5 neomycin phosphotransferase bacterial promoter of transposon TN5 hygromycin phosphotransferase plasmid replication origin.pBR322 poisoning-sequence, pBR322 sequence inhibiting SV40 replication origin of SV40 replication coinciding with early and late promoters bp enhancer of SV40 early promoter enhancer of human cytomegalovirus (HCMV) major common early gene promoter/mRNA start site of murine cytomegalovirus (MCMC) major abrupt early gene Rous sarcoma virus LTR (promoter) position 5’ m7Gp cap eukaryotic mRNA polyadenylation site mRNA binding donor site, 5’ end of intron binding acceptor site, 3’ end of intron bacterial alkaline phosphatase calf intestinal phosphatase
Sau3a mesto, ki rezultira iz po.vezave-. BamHI in Bg lil mesta mutirano Seal mesto mesto x restrikcijskega encima·, ki se nahaja v smeri urnega kazalca od ySau3a site resulting from the ligation of BamHI and Bg lil sites mutated Seal site restriction enzyme site x located clockwise from y
- 42 Eksperimentalni del- 42 Experimental part
Primer 1: Vpeljava Seal mesta na vez med strukturami krigle in encimsko domeno v humani t-PA cDNAExample 1: Introduction of a Seal site at the junction between the mug structures and the enzyme domain in human t-PA cDNA
Princip, uporabljen za konstrukcijo himeričnih in hibridnih molekul, ki vsebujejo domene obeh t-PA in u-PA, sestoji iz priprave zaželjenih restrikcijskih fragmentov, ki izhajajo iz poedinih klonov, njihovega ponovnega sestavljanja v raztopini, nato kloniranja izhajajočih konstrukcij. Po kloniranju preverimo strukturo himeričnih molekul z restrikcijskim mapiranjem in analizo DNA zaporedja.The principle used for the construction of chimeric and hybrid molecules containing both t-PA and u-PA domains consists of preparing the desired restriction fragments derived from individual clones, reassembling them in solution, and then cloning the resulting constructs. After cloning, the structure of the chimeric molecules is verified by restriction mapping and DNA sequence analysis.
Da dobimo hibridne molekule obeh t-Pa in u-PA cDNA, cepimo na vezeh med poedinimi strukturami kringle in encimskimi domenami. To dosežemo z u-Pa, da izvedemo delno prebavo z restrikcijsko endonukleazo Mstl, ki loči nekatalitično domeno od encimske domene in priključenih zaporedij na 3’koncu. Nobeno primerljivo, uporabno, potencial no cepitveno mesto ni prisotno v t-PA, in eno zatorej vpeljemo, kot je opisano spodaj:To obtain hybrid molecules of both t-PA and u-PA cDNA, we cleave at the junctions between the individual kringle structures and the enzyme domains. This is achieved by using u-PA to perform a partial digestion with the restriction endonuclease MstI, which separates the non-catalytic domain from the enzyme domain and the 3'-terminal flanking sequences. No comparable, useful, potential cleavage site is present in t-PA, and one is therefore introduced as described below:
A) Konstrukcija plazmida pEcoO.47A,ScaI (glej sl. 5)A) Construction of plasmid pEcoO.47A,ScaI (see Fig. 5)
V tej konstrukciji edino Seal mesto (AGTACT) na nuUAeotidni legi 940-945 t-PA cDNA razgradimo (AGTACT AGTATT) in vpeljemo drugo Seal mesto na nukleotidnih legah (963-968 (T£CACC -+ AGTACT) na 3?koncu kringle 2» (primerjaj sl. 1 in 2). Kodiranje nobene od amino kislin ni prizadeto s temi spremembami.In this construction, the only Seal site (AGTACT) at nucleotide positions 940-945 of the t-PA cDNA is degraded (AGTACT AGTATT) and a second Seal site is introduced at nucleotide positions (963-968 (T£CACC -+ AGTACT) at the 3' end of kringle 2" (compare Figs. 1 and 2). The coding of any amino acids is not affected by these changes.
- 43 Vse restrikcijske prebave izvršimo v smislu navodil proizvajalcev (New England Biolabs, Bethesda Research Labs) in nastale prebavke analiziramo z elektroforezo na 3,5 % poliakrilamidnem gelu. Gel barvamo z homidijev bromidom (1,0 ^pg/ml) in predočimo z ultravijolično svetlobo. Primeren pas izrežemo in elektroeluiramo v 0,5 χ TBE (1 χ TBE = 90 mM Tris-borata, pH 8,3, 2,5 mM EDTA). Elektroeluiran material apliciramo na Elutip-d kolono (Schleicher and Schuell), vezano DNA eluiramo v močno slanem in preeipitiramo z dodatkom etanola. Usedlino speremo v etanolu, posušimo in raztopimo v vodi .- 43 All restriction digests were performed according to the manufacturers' instructions (New England Biolabs, Bethesda Research Labs) and the resulting digests were analyzed by electrophoresis on a 3.5% polyacrylamide gel. The gel was stained with homidium bromide (1.0 µg/ml) and visualized under ultraviolet light. The appropriate band was excised and electroeluted in 0.5 χ TBE (1 χ TBE = 90 mM Tris-borate, pH 8.3, 2.5 mM EDTA). The electroeluted material was applied to an Elutip-d column (Schleicher and Schuell), the bound DNA was eluted in high-salt solution and precipitated with the addition of ethanol. The precipitate was washed in ethanol, dried and dissolved in water.
Plazmid pW349F (evropska patentna prijava št. 143,081), ki vsebuje humano t-PA cDNA (sintetizirana iz mRNA, izolirane iz HeLaS3 celic in klonirana v Pstl mestu plazmida pBR322) se prebavi z EcoRI in fragment 470 baznih parov (bp) izoliramo (prim. sl.2). S prebavo 470 bp EcoRI fragmenta s Seal oziroma Haelll dobimo 150 bp EcoRI, Seal in 290 bp EcoRI, Haelll fragmente. Dve vlakni 470 bp EcoRI fragmenta ločimo z denaturiranjem DNA v DMSO pufru (30 % DMSO, 1 mM EDTA, 0,5 % ksilen cianol, 0,05 % bromfenol modro) in izvedemo elektroforezo na 5 % poliakrilamidnem gelu v 0,5 x TBE pri 8 voltih na centrimeter /Maniatis et al., Molecular Clonlng, A Laboratory Manual, Cold Spring Harbor Laboratory; 1982/. Ločeni vlakni pridobimo z elektroelucijo, ki ji sledi precipitacija z etanolom. 31-mer deoksioligonukleotid (ki ima vključenih 5 zaželjenih nukleotidnih sprememb, primerjaj sl. 5) sintetiziramo z uporabo fosfotriesterske - 32 metode. Petdeset pmolov 31-mer označimo s P na 5’koncu v reakcije, ki vsebuje 1 x kinazni pufer (10 x kinazni pufer - 0,5 M Tris.HCl, pH 7,5, 0,1 M MgCl^, 50 mM DTT, 1 mM spermidin, 1 mM EDTA), 30 ji Ci /oD ^^P/ATP (Amersham, 3000 Ci/mmol) in 10 enot T^ polinukleotid kinaze (Bethesda Research Labs.). Reakcijo inkubiramo pri 37°C 30 minut, sledi dodatek 1 jil 10 mM ATP, 10 enot T^ kinaze in nadalje 30 minut inkubacije pri 37°C. Reakcijo končamo s segrevanjem pri 68°C 10 minut. Označeni 31-mer, čigar zaporedje je tisto ne-prepisanega vlakna, preizkušamo v ”dot blot analizi /izvedena v smislu Zoller and Smith, Nucl. Acids Res., 10, 6487-6500 (1982); razen da sta predhodna hibridizacija in hibridizacija izvedeni pri 50°C in izpiranje pri 60°C/ za določitev, kateri od dveh vlaken se z njim hibridizira, t.j. predstavlja prepisano vlakno. Štiri DNA pomešamo skupaj v 20 renaturaeijske reakcije, ki sestoji iz 0,3 pmolov prepisanega vlakna, 2 pmolov vsakega od 150 bp EcoRI, Seal in 290 bp, EcoRI, HaelIT fragmentov, 25 pmolov fosforilirenega 31-mer in 1 x renat. pufra (5 x renat. pufer = 0,5 m NaCl, 32,5 mM Tris.HCl pH 7,5, 40 mM MgCl2 in 5 mM -merkaptoetanol). Mešanico inkubiramo pri 100°C 3 minute, 30°C 30 minut, 4°C 30 minut in nato na ledu 10 minut, sledeč temu, dodamo 400 enot DNA ligaze (New England Biolabs) in reakcijo inkubiramo pri 12,5°C čez noč. 470 bp nenaturirani fragment pridobimo iz 3,5 % poliakrilamidnega gela, kot je opisano zgoraj, in vežemo na z EcoRI prebavljeno in defosforilizirano pBR322 DNA (New England Biolabs) v 50 mM Tris.HCl pH 7,5, 10 mM MgClp, 10 mM DTT,1mMPlasmid pW349F (European Patent Application No. 143,081), containing human t-PA cDNA (synthesized from mRNA isolated from HeLaS3 cells and cloned into the PstI site of plasmid pBR322), was digested with EcoRI and a 470 base pair (bp) fragment was isolated (cf. Fig. 2). Digestion of the 470 bp EcoRI fragment with Sial and HaelII, respectively, yielded 150 bp EcoRI, Sial and 290 bp EcoRI, HaelII fragments. The two strands of the 470 bp EcoRI fragment were separated by denaturing the DNA in DMSO buffer (30% DMSO, 1 mM EDTA, 0.5% xylene cyanol, 0.05% bromophenol blue) and electrophoresed on a 5% polyacrylamide gel in 0.5 x TBE at 8 volts per centimeter /Maniatis et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; 1982/. Separated fibers were obtained by electroelution followed by ethanol precipitation. A 31-mer deoxyoligonucleotide (incorporating the 5 desired nucleotide changes, cf. Fig. 5) was synthesized using the phosphotriester-32 method. Fifty pmol of the 31-mer was labeled with P at the 5' end in a reaction containing 1 x kinase buffer (10 x kinase buffer - 0.5 M Tris.HCl, pH 7.5, 0.1 M MgCl^, 50 mM DTT, 1 mM spermidine, 1 mM EDTA), 30 µCi/oD ^^P/ATP (Amersham, 3000 Ci/mmol) and 10 units of T^ polynucleotide kinase (Bethesda Research Labs.). The reaction is incubated at 37°C for 30 minutes, followed by the addition of 1 µl of 10 mM ATP, 10 units of T^ kinase and a further 30 minutes of incubation at 37°C. The reaction is terminated by heating at 68°C for 10 minutes. The labeled 31-mer, whose sequence is that of the non-transcribed strand, is tested in a dot blot analysis (performed according to Zoller and Smith, Nucl. Acids Res., 10, 6487-6500 (1982); except that the prehybridization and hybridization are performed at 50°C and the wash at 60°C) to determine which of the two strands hybridizes to it, i.e. represents the transcribed strand. The four DNAs are mixed together in a 20 renaturation reaction consisting of 0.3 pmoles of the transcribed strand, 2 pmoles each of the 150 bp EcoRI, Sial and 290 bp, EcoRI, HaelIT fragments, 25 pmoles of phosphorylated 31-mer and 1 x renaturation buffer (5 x renaturation buffer = 0.5 m NaCl, 32.5 mM Tris.HCl pH 7.5, 40 mM MgCl2 and 5 mM -mercaptoethanol). The mixture was incubated at 100°C for 3 min, 30°C for 30 min, 4°C for 30 min and then on ice for 10 min, following which 400 units of DNA ligase (New England Biolabs) were added and the reaction incubated at 12.5°C overnight. The 470 bp non-denatured fragment was recovered from a 3.5% polyacrylamide gel as described above and ligated to EcoRI digested and dephosphorylated pBR322 DNA (New England Biolabs) in 50 mM Tris.HCl pH 7.5, 10 mM MgCl, 10 mM DTT, 1 mM
ATP, 1 mM spermidina, 0,1 mg/ml govejega serum albumina z inkubacijo pri 12°C čez noč. Mešanice za vezanje uporabljamo, da transformiramo primeren E.coli soj HB101 (Maniatis et al., supra). Ampicilin - odporne kolonije ločimo na L-agarju, ki vsebuje 50 ^ig/ml ampicilina, in kolonije, ki vsebujejo 470 bp fragment identificiramo s hibridizacijo kolonije z uporabo 31-mer kot preizkušanca /d. Woods, Focus _6, 1-3 ( 1984)/. Plazmid DNA izoliramo iz posameznih hibridizacijskih kolonij v majhni meri /Holmes et al., Analyt. Biochem. 114, 193-197 (1981)/ in ustvarjenje novega Seal mesta potrdimo s kombinirano EcoRI in Seal prebavo. Da zagotovimo čistost, uporabljamo plazmid DNA iz pozitivnih kolonij v drugem krogu transformacije E.coli HB101. Pripravo plazmida v veliki meri napravimo iz ene takih pozitivnih kolonij druge generacije /Katz et al., J. Bacteriol.ATP, 1 mM spermidine, 0.1 mg/ml bovine serum albumin with incubation at 12°C overnight. The ligation mixtures were used to transform the appropriate E. coli strain HB101 (Maniatis et al., supra). Ampicillin-resistant colonies were isolated on L-agar containing 50 µg/ml ampicillin, and colonies containing the 470 bp fragment were identified by colony hybridization using a 31-mer as a probe /d. Woods, Focus _6, 1-3 (1984)/. Plasmid DNA was isolated from individual hybridization colonies in small amounts /Holmes et al., Analyt. Biochem. 114, 193-197 (1981)/ and the creation of a new Seal site was confirmed by combined EcoRI and Seal digestion. To ensure purity, plasmid DNA from positive colonies was used in a second round of transformation of E. coli HB101. Plasmid preparation is largely done from one such positive second generation colony /Katz et al., J. Bacteriol.
14, 577-591 ( 1 973); Biochemistry J_6, 1677-1683 ( 1977) in uničevanje prvotnega Seal mesta in ustvarjanje novega Seal mesta potrdimo z analizo DNA zaporedja z uporabo metode Maxam and Gilbert /Methods Enzym. 65, 499-560 ( 1980)/. Ta plazmid označimo pEcoO.47A.ScaI.14, 577-591 (1973); Biochemistry J_6, 1677-1683 (1977) and the destruction of the original Seal site and the creation of a new Seal site were confirmed by DNA sequence analysis using the method of Maxam and Gilbert /Methods Enzym. 65, 499-560 (1980)/. This plasmid was designated pEcoO.47A.ScaI.
B) Rekonstrukcija humanega t-PA z mutiranim Seal mestom (glej sl. 6)B) Reconstruction of human t-PA with a mutated Seal site (see Fig. 6)
V tej konstrukciji 470 bp EcoRI fragment, prisoten v divji vrsti humanega t-PA,zamenjamo s 470 bp EcoRI fragmentom, ki vsebuje mutirano Seal mesto. Plazmid pW349F, ki vsebuje humano t-PA cDNA (glej zgoraj) prebavimo s Clal in nastale lepljive konce napravimo tope z dodatkom 50 jim vsakega od dCTP, dGTP in 10 enot DNA polimeraze I, Klenow fragment (Boehringer, Mannheim). Reakcijo inkubiramo pri sobni tempera46 turi 30 minut ter sledi ekstrakcija s fenolom in etrom in precipitacija z etanolom. Usedlino raztopimo v vodi, prebavimo z EcoRI in Seal in 1,5 kb EcoRI, Seal in 4,3 kb Clal (s topimi konci), EcoRI fragmenta izoliramo. Ta dva fragmenta pomešamo s 470 bp fragmentom, ki ga pridobimo iz plazmida pEcoO.473. Seal po ECoRI prebavi in vežemo, kot je opisano zgoraj, pri 12°C čez noč. Primerne E.coli HB101 celice transformiramo z mešanico za vezanje in tetraciklin-odporne kolonije ločimo na L-agarju, ki vsebuje 12,5 ^ig/ml tetraciklina.In this construction, the 470 bp EcoRI fragment present in wild-type human t-PA is replaced by a 470 bp EcoRI fragment containing a mutated Seal site. Plasmid pW349F containing the human t-PA cDNA (see above) is digested with ClaI and the resulting sticky ends are made blunt by the addition of 50 µm each of dCTP, dGTP and 10 units of DNA polymerase I, Klenow fragment (Boehringer, Mannheim). The reaction is incubated at room temperature for 30 minutes followed by extraction with phenol and ether and precipitation with ethanol. The pellet is dissolved in water, digested with EcoRI and Seal and the 1.5 kb EcoRI, Seal and 4.3 kb ClA (blunted-end), EcoRI fragment is isolated. These two fragments are mixed with the 470 bp fragment obtained from plasmid pEcoO.473. Seal after ECoRI digestion and ligate as described above at 12°C overnight. Suitable E. coli HB101 cells are transformed with the ligation mixture and tetracycline-resistant colonies are isolated on L-agar containing 12.5 µg/ml tetracycline.
Kolonije, ki vsebujejo 470 bp mutiran fragment,identificiramo s hibridizacijo kolonije ob uporabi predhodno opisanega 31-mer kot preizkušanca. Pripravimo DNA iz minilizatov posameznih pozitivno hibridiziranih kolonij in potrdimo natančno naravo konstrukcije tako, da napravimo primerne restrikcijske prebavke (digests). En tak plazmid z željenimi spremembami imenujemo ph.tPA/SScal.Colonies containing the 470 bp mutated fragment are identified by colony hybridization using the previously described 31-mer as a probe. DNA is prepared from minilysates of individual positively hybridized colonies and the exact nature of the construct is confirmed by making appropriate restriction digests. One such plasmid with the desired changes is designated ph.tPA/SScal.
Primer. 2: Konstrukcija u-PA/t-PA.hibridne molekule; plazmid pUNC.tc (glej sl. 7)Example 2: Construction of u-PA/t-PA hybrid molecule; plasmid pUNC.tc (see Fig. 7)
Ta konstrukcija je hibrid med nekatalitičnem območjem u-PA (ki vsebuje 5’nekodirajoče območje, signalno, rastni faktor in kringle zaporedje) in katalitično ali encimsko domeno humanega t-PA.This construct is a hybrid between the non-catalytic region of u-PA (containing the 5’ non-coding region, signal, growth factor and kringle sequences) and the catalytic or enzymatic domain of human t-PA.
Urokinazno cDNA pripravimo iz mRNA, dobljeno iz humanih Hep3 celic /cf. T. Maniatis et al., Molecular Cloning (1982), str. 188-246/. 1,3 kb Smal-BamHI fragment in 1 kbUrokinase cDNA was prepared from mRNA obtained from human Hep3 cells /cf. T. Maniatis et al., Molecular Cloning (1982), pp. 188-246/. A 1.3 kb SmaI-BamHI fragment and a 1 kb
BamHI-EcoRI fragment u-PA cDNA kloniramo v Smal, EcoRI mestih pUN121 /B.Nilsson et al., Nucl. Acids Res. 11 , 8019-8030 (1983)/, da dobimo plazmid pcUK176. Oris restrikcijske endonukleaze vključka humanega u-PA cDNA je prikazan v sl. 4. Zaporedje nukleotidov in izvedeno zaporedje amino kislin u-PA vključka je dano v sl. 3.The BamHI-EcoRI fragment of the u-PA cDNA was cloned into the SmaI, EcoRI sites of pUN121 (B. Nilsson et al., Nucl. Acids Res. 11, 8019-8030 (1983)) to yield plasmid pcUK176. The restriction endonuclease outline of the human u-PA cDNA insert is shown in Fig. 4. The nucleotide sequence and deduced amino acid sequence of the u-PA insert are given in Fig. 3.
Plazmid pcUK176 se prebavi z Xmal (primerjaj sl. 4; Xmal je izoshizomer Smal) in Mstl in izoliramo 521 bp fragment. Restrikcijski encim Mstl prepozna DNA zaporedje TGC GCA (puščici kažeta mesto cepitve) in naredi po prebavi topa konca; ta encim zato veže u-PA cDNA pri nukleotidih 520-525, t.j. ravno za zadnjim cisteinskim preostankom (amino kislina 131), ki obsega kringle /Holmes et al., Bioteohnology _3, 923-929 ( 1985)/,' in tako čisto loči zaporedja za kodiranje nekatalitičnega in katalitičnega območja.Plasmid pcUK176 is digested with XmaI (compare Fig. 4; XmaI is an isoschizomer of SmaI) and MstI, and a 521 bp fragment is isolated. The restriction enzyme MstI recognizes the DNA sequence TGC GCA (arrows indicate the cleavage site) and makes blunt ends after digestion; this enzyme therefore binds the u-PA cDNA at nucleotides 520-525, i.e. just after the last cysteine residue (amino acid 131) comprising the kringle (Holmes et al., Bioteohnology _3, 923-929 ( 1985)),' and thus clearly separates the sequences coding for the non-catalytic and catalytic regions.
Plazmid ph.tPAzsScal se prebavi s Seal in Hindlll (Hind III je prisoten v vektorju) in pridobimo 1,8 kb fragment. Restrikcijski encim Seal prepozna DNA zaporedje AGT ACT (puščici kažeta mesto cepitve) in da tudi topa konca po prebavi. Seal bo rezal ph.tPAAScal DNA po serinskem preostanku 262 /1 amino kislina po zadnjem cisteinu krigle 2; Pennica et al., Nature 301, 214-221 (1983)/, zato ločuje nekatalitično in katalitično domeno.Plasmid ph.tPAzsScal is digested with Seal and HindIII (Hind III is present in the vector) and a 1.8 kb fragment is obtained. The restriction enzyme Seal recognizes the DNA sequence AGT ACT (arrows indicate the cleavage site) and also blunts the ends after digestion. Seal will cut ph.tPAAScal DNA at serine residue 262 (1 amino acid after the last cysteine of loop 2; Pennica et al., Nature 301, 214-221 (1983)), thus separating the noncatalytic and catalytic domains.
Dva fragmenta pomešamo in vežemo na Xmal, Hindlll cepljeno pUCl8 vektorsko DNA. Po transformaciji E.coli HB101, kolonije, ki imajo pravilen vključek, identificiramo s hibridizacijo kolonij z uporabo 2,0 kb Bglll fragmenta humanega tPA (primerjaj sl. 2) kot preizkušanca, ki je označen po metodiThe two fragments are mixed and ligated to XmaI, HindIII cleaved pUC18 vector DNA. After transformation of E. coli HB101, colonies having the correct insert are identified by colony hybridization using the 2.0 kb BglII fragment of human tPA (compare Fig. 2) as a probe, which is labeled according to the method
Primer 3: Konstrukcija t-PA/u-PA hibridne molekule; plazmid ptNC.UC (primerjaj sl. 8)Example 3: Construction of a t-PA/u-PA hybrid molecule; plasmid ptNC.UC (compare Fig. 8)
Ta konstrukcija je ravno nasprotje pUNC.tc v tem, da je nekatalitično območje ph.tPATScal (ki vsebuje 5’nekodirajoče območje, vodenih, finger, rastni faktor, kringle 1 in kringle 2 domene) zlito s katalitično domeno humanega u-PA. Plazmid ph.tPAAScal se prebavi s SacI in Seal (primerjaj sl. 8) in fragment okoli 1,0 kb izoliramo. Plazmid pcUK176 se najprej prebavi z BamHI in nato delno cepi z Mstl in pridobimo fragment z okoli 800 bp. Takoj sledi, da BamHI prebavek režemo z EcoRI in fragment okoli 1,0 kb izoliramo. Te tri fragmente pomešamo z pUC19 vektor jem, prebavljenim s SacI, EcoRI in povežemo. E.coli HB101 transformiramo z mešanico za vezanje, in kolonije, ki imajo pravilen vključek, identificiramo s hibridizacijo kolonije z uporabo istega 20 kb Bglll preizkušanca, kot je opisano zgoraj. DNA zaporedje na zvezi t-PA in u-PA DNA potrdimo z analizo DNA zaporedja. En pravilen klon imenujemo ptNC.UC.This construct is the exact opposite of pUNC.tc in that the non-catalytic region of ph.tPATScal (containing the 5’ non-coding region, guide, finger, growth factor, kringle 1 and kringle 2 domains) is fused to the catalytic domain of human u-PA. Plasmid ph.tPAAScal is digested with SacI and Seal (compare Fig. 8) and a fragment of about 1.0 kb is isolated. Plasmid pcUK176 is first digested with BamHI and then partially cleaved with MstI to obtain a fragment of about 800 bp. The BamHI digest is then cut with EcoRI and a fragment of about 1.0 kb is isolated. These three fragments are mixed with the pUC19 vector digested with SacI, EcoRI and ligated. E. coli HB101 was transformed with the ligation mix, and colonies having the correct insert were identified by colony hybridization using the same 20 kb BglII probe as described above. The DNA sequence at the junction of t-PA and u-PA DNA was confirmed by DNA sequence analysis. One correct clone was designated ptNC.UC.
Primer 4: Konstrukcija ekspresijskega vektorja za uporabo v celicah sesalcevExample 4: Construction of an expression vector for use in mammalian cells
A) Pretvorba HgiAI mesta v t-PA cDNA v HindZII mestoA) Conversion of the HgiAI site in the t-PA cDNA to the HindZII site
To dosežemo v petih stopnjah (sl. 9).This is achieved in five stages (Fig. 9).
- 49 Plazmid pW349E (evropska patentna prijava št. 143,081 ) delno cepimo z restrikcijskim encimom HgiAI pri inkubaciji 20 ^jg/ml DNA 1 uro pri 37°C z 12 U/ml encima v pufru, ki ga priporoča proizvajalec (Bethesda Research Laboratories), razen da je dopolnjen z 10 ^ig/ml homidijevega bromida, zadrži drugo rezanje plazmida. Plazmid DNA v linearni obliki nato položimo na 0,8% agarozni gel v TBE pufru /TBE: 89 mM Tris-borat, pH 8,9, ki vsebuje 1 mM EDTA), elektroforetično eluiramo v istem pufru, dvakrat ekstrahiramo z fenolom, dvakrat s kloroformom in končno pre cipitiramo z alkoholom pri -20°C po dodatku 0,1 vol.- 49 Plasmid pW349E (European Patent Application No. 143,081) was partially cleaved with the restriction enzyme HgiAI by incubating 20 µg/ml DNA for 1 hour at 37°C with 12 U/ml of the enzyme in the buffer recommended by the manufacturer (Bethesda Research Laboratories), except that it was supplemented with 10 µg/ml of homidium bromide, which inhibits the second cleavage of the plasmid. The plasmid DNA in linear form was then loaded onto a 0.8% agarose gel in TBE buffer (TBE: 89 mM Tris-borate, pH 8.9, containing 1 mM EDTA), electrophoretically eluted in the same buffer, extracted twice with phenol, twice with chloroform and finally precipitated with alcohol at -20°C after the addition of 0.1 vol.
M natrijevega acetata, pH 5,2. Usedlo DNA raztopimo pri 0,2 mg/ml v TE (TE : 10 mM Tris-HCl pH 7,2 z 0,1 mM EDTA).M sodium acetate, pH 5.2. The precipitated DNA was dissolved at 0.2 mg/ml in TE (TE: 10 mM Tris-HCl pH 7.2 with 0.1 mM EDTA).
^il DNA v linearni obliki nato inkubiramo 30 minut pri 37°C s 15 U T4 DNA polimeraze v ligaznem pufru /33 mM Tris-acetat (pH 7,9), 66 mM kalijev acetat, 10 mM magnezijev acetat, 0,5 mM ditiotreitol in 0,1 mg/ml goveji serum albumin/; sledi segrevanje 10 min. pri 60°C, da se encim inaktivira.The linearized DNA was then incubated for 30 minutes at 37°C with 15 U of T4 DNA polymerase in ligase buffer (33 mM Tris-acetate (pH 7.9), 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM dithiothreitol, and 0.1 mg/ml bovine serum albumin); followed by heating for 10 minutes at 60°C to inactivate the enzyme.
Namen te inkubacije je uporaba eksonukleolitične aktivnosti T4 polimeraze za odstranitev preostalih ven štrlečih štirih nukleotidov po prebavi s HgiAI, da dobimo DNA molekule s topimi konci.The purpose of this incubation is to use the exonucleolytic activity of T4 polymerase to remove the remaining four overhanging nucleotides after digestion with HgiAI to obtain DNA molecules with blunt ends.
Zato, da zvežemo Hindlll povezovalce (CAAGCTTG) z DNA s topim koncem, dodamo 6 ^il (300 ng) kinaznih povezovalcev h gornji raztopini z 4 ^pl 10 mM ATP in 3 ^1 T4 DNA ligaze (New England Biolabs, 400 U/^jl), sledi 16-urna inkubacija pri 16°C. Zvezanje je končano s segrevanjem mešanice 10 min. pri 68°C, po katerem se DNA prebavi s Hindlll in Bglll, t.j. 15y 1 (135 U) Hindlll dodamo 1,5 4M NaCl, 0,2 ^1 in 1M MgCl2 in 11 ^ιΐ 1 mg/ml govejega serum albumina, inkubiramo pri 37°C 1 uro, sledi dodatek 40 U Bglll, sledi še ena ura inkubacije pri 37°C. Nastali fragment s 177 baznimi pari čistimo na 6 % poliakrilamidnem gelu, tekočem v TBE, eluiramo v TNE (TNE: 10 mM Tris-HCl pH 8,8, ki vsebuje 100 mM NaCl in 1 mM EDTA), absorbiramo na DEAE celulozo (Whatman DE52), eluiramo z 1M NaCl v TNE, razredčimo s 4 volumni vode, precipitiramo pri -20°C po dodatku 2,5 volumnov etanola in končno raztopimo v 17 TE (TE: 10 mM Tris-HCl pH 8,0, ki vsebuje 1 mM EDTA).To ligate the HindIII linkers (CAAGCTTG) to blunt-ended DNA, 6 µl (300 ng) of kinase linkers were added to the above solution with 4 µl of 10 mM ATP and 3 µl of T4 DNA ligase (New England Biolabs, 400 U/µl), followed by a 16-hour incubation at 16°C. Ligation was terminated by heating the mixture for 10 min at 68°C, after which the DNA was digested with HindIII and BglII, i.e., 15 µl (135 U) of HindIII was added with 1.5 µl of 4M NaCl, 0.2 µl of 1M MgCl2,and 11 µl of 1 mg/ml bovine serum albumin, incubated at 37°C for 1 hour, followed by the addition of 40 U of BglII, followed by another hour of incubation at 37°C. The resulting 177 base pair fragment was purified on a 6% polyacrylamide gel running in TBE, eluted in TNE (TNE: 10 mM Tris-HCl pH 8.8, containing 100 mM NaCl and 1 mM EDTA), absorbed onto DEAE cellulose (Whatman DE52), eluted with 1M NaCl in TNE, diluted with 4 volumes of water, precipitated at -20°C after addition of 2.5 volumes of ethanol and finally dissolved in 17 TE (TE: 10 mM Tris-HCl pH 8.0, containing 1 mM EDTA).
Plazmid pRSVneo je izvedenka plazmida pSV2neo /P.J. Southern and P. Berg, J. Mol. Appl. Genet. _1_> 327-341 (1982)/ v katerem je SV40-izveden PvuII-Hindlll fragment nadomeščen s PvuII-Hindlll fragmentom, ki vsebuje LTR promotor iz virusa Rousovega sarkoma, na isti način, kot je bil konstruiran pRSVcat iz pSV2cat /C.M. Gorman et al., Proč. Natl. Acad. Sci USA'29, 6777-6781 (1982)/. 5 fg tega plazmida se reže v 50 ^1 volumnu z 24 U Bglll v smislu proizvajalca. Po 1 uri inkubaciji pri 37°C dodamo 40 U Hindlll in inkubacijo nadaljujemo 1,5 ure, po tem velik 5,4 kb fragment čistimo, kot je opisano zgoraj.Plasmid pRSVneo is a variant of plasmid pSV2neo (P.J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341 (1982)) in which the SV40-derived PvuII-HindIII fragment has been replaced by a PvuII-HindIII fragment containing the LTR promoter from Rous sarcoma virus, in the same manner as pRSVcat was constructed from pSV2cat (C.M. Gorman et al., Proc. Natl. Acad. Sci USA 29:6777-6781 (1982)). 5 fg of this plasmid is digested in a 50 µl volume with 24 U BglII according to the manufacturer's instructions. After 1 hour of incubation at 37°C, 40 U HindIII is added and the incubation is continued for 1.5 hours, after which the large 5.4 kb fragment is purified as described above.
očiščenega 177 bp fragmenta vežemo 18 ur pri 16°C na 2 f 1 (20 ng) pRSVneo fragmenta ob uporabi 0,25 jil (100 U) T4 ligaze v celotnem volumnu 22 ligaznega pufra, po tem je plazmid DNA uporaben, da transformira E.coli v smislu D. Hanahan /J. Mol. Biol. 166, 557-580 ( 1983)/. Iz izhajajočih ampicilin-odpornih sojev izberemo enega, ki vsebuje plazmid,označen ptPAL z 177 bp Hindlll-Bglll fragmentom, kot je razvidno pri restrikcijski analizi. 0,1 fg tega plazmida tešemo v 16 ^il s 16 U BgllT, kot priporoča proizvajalec 1,5 ure pri 37°C. K tej raztopini nato dodamo 20 U goveje črevesne alkalne fosfataze (Boehringer Mannheim) in nadaljujemo inkubacijo še 30 min., nakar DNA ekstrahiramo dvakrat s fenolom, dvakrat s kloroformom in precipitiramo po dodatku 0,1 volumna 3,0 M natrijevega acetata pH 5,2 in 0,6 volumna izopropanola, raztopimo v TE, nadalje čistimo z agarozno gel elektroforezo, kot je opisano zgoraj, dvakrat ekstrahiramo s fenolom, dvakrat s kloroformom, precipitiramo pri -20°C po dodatku 2,5 volumna etanola in 0,1 vol. 3M natrijevega acetata pH 5,2 in končno raztopimo v 30 ^il TE. 2,1 kb tPA Bglll fragment nato izrežemo iz 5 pW349F v 25 reakcije z uporabo 20 U Bglll 2 uri pri 37°C, čistimo na 0,8 % agaroznem gelu, eluiramo z elektroforezo, kot je opisano zgoraj, dvakrat ekstrahiramo s fenolom, dvakrat s kloroformom, precipitiramo pri -20°C po dodatku 2,5 volumnov etanola in 0,1 vol. 3 M natrijevega acetata pH 5,2 in raztopimo pri koncentraciji 8 ng/^il v TE. 1 t-PA fragmenta nato vežemo v 10 ^il reakcije na 7,5 ng Bglll rez vektorske DNA z uporabo 100 U T4 ligaze (Biolabs) 17 ur pri 16°C in nato transformiramo v E.ooli. Eden od izhajajočih klonov, označen pD02, vsebuje t-PA Bglll fragment, vključen na tak način, da vsebuje plazmid kontinuirno odprt čitalni okvir za humani t-PA.The purified 177 bp fragment is ligated for 18 hours at 16°C to 2 µl (20 ng) of the pRSVneo fragment using 0.25 µl (100 U) of T4 ligase in a total volume of 22 µl of ligase buffer, after which the plasmid DNA is used to transform E. coli according to D. Hanahan /J. Mol. Biol. 166, 557-580 (1983)/. From the resulting ampicillin-resistant strains, one is selected which contains a plasmid designated ptPAL with a 177 bp HindIII-BglII fragment, as shown by restriction analysis. 0.1 µg of this plasmid is weighed into 16 µl with 16 U of BglII, as recommended by the manufacturer, for 1.5 hours at 37°C. To this solution was then added 20 U of bovine intestinal alkaline phosphatase (Boehringer Mannheim) and incubation continued for another 30 min., after which the DNA was extracted twice with phenol, twice with chloroform and precipitated after the addition of 0.1 volume of 3.0 M sodium acetate pH 5.2 and 0.6 volume of isopropanol, dissolved in TE, further purified by agarose gel electrophoresis as described above, extracted twice with phenol, twice with chloroform, precipitated at -20°C after the addition of 2.5 volumes of ethanol and 0.1 vol. of 3M sodium acetate pH 5.2 and finally dissolved in 30 µl of TE. The 2.1 kb tPA BglII fragment was then excised from 5 pW349F in 25 reactions using 20 U BglII for 2 hours at 37°C, purified on a 0.8% agarose gel, eluted by electrophoresis as described above, extracted twice with phenol, twice with chloroform, precipitated at -20°C after addition of 2.5 volumes of ethanol and 0.1 vol. 3 M sodium acetate pH 5.2 and dissolved at a concentration of 8 ng/µl in TE. 1 t-PA fragment was then ligated in 10 µl reactions to 7.5 ng of BglII cut vector DNA using 100 U T4 ligase (Biolabs) for 17 hours at 16°C and then transformed into E. coli. One of the resulting clones, designated pD02, contains the t-PA BglII fragment, inserted in such a way that the plasmid contains a contiguous open reading frame for human t-PA.
B) Kombinacija t-PA cDNA s fragmentom beta-globinaB) Combination of t-PA cDNA with a beta-globin fragment
Plazmid pD010 (sl. 10) konstruiramo s povezavo trehPlasmid pD010 (Fig. 10) is constructed by linking three
DNA fragmentov: (i) 2,1 kb fragment, ki se začenja s Hindlll mestom in se končuje s Bglll mestom, ter vsebuje celotno t-PA kodirno zaporedje izolirano iz agaroznega gela, na katerega naložimo 10 pD02 DNA delno rezano z Bglll in popolnoma s Hindlll. (ii) pUB je plazmid, ki vsebuje zajčev gen beta globina /A. Van 0oyen et al., Science 206, 337 (1979)/, subkloniran kot Bglll delni prebavek v BamHI mestu plazmida pUC9 /J. Vieira and J. Messing, Gene 19, 259-268 (1982); ibid. 19, 269-276 (1982)/. Iz tega plazmida izrežemo 1.2 kb BamHI-Hindlll fragment, ki vsebuje drugi intron in poliadenilacijsko mesto in ga čistimo z agarozno gel elektroforezo. (iii) Vektor pD01 je vgrajen v nasprotni smeri urnega kazalca, iz Hindlll mesta (sl. 10) Hindlll-AccI fragmenta pBR322, kateri vključuje začetek podvojevanja, 0,3 kb fragmenta, ki vsebuje povečevalnik humanega citomegalovirusa (HCMV), ki se končuje v sintetičnem Xbal mestu in mu sledi druga kopija tega povečevalnika, pritrjenega na homologni promotor, ki se končuje na sintetičnem Hindlll mestu. Ta vektor DNA režemo s Hindlll in 6,3 kb linearni plazmid čistimo z agarozno gel elektroforezo.DNA fragments: (i) a 2.1 kb fragment starting at the HindIII site and ending at the BglIII site, containing the entire t-PA coding sequence, isolated from an agarose gel onto which 10 pD02 DNA cut partially with BglIII and completely with HindIII was loaded. (ii) pUB is a plasmid containing the rabbit beta globin gene /A. Van Ooyen et al., Science 206, 337 (1979)/, subcloned as a BglIII partial digest in the BamHI site of plasmid pUC9 /J. Vieira and J. Messing, Gene 19, 259-268 (1982); ibid. 19, 269-276 (1982)/. A 1.2 kb BamHI-HindIII fragment containing the second intron and the polyadenylation site was excised from this plasmid and purified by agarose gel electrophoresis. (iii) The pD01 vector is inserted counterclockwise from the HindIII site (Fig. 10) of the HindIII-AccI fragment of pBR322, which includes the origin of replication, a 0.3 kb fragment containing the human cytomegalovirus (HCMV) enhancer, ending in a synthetic XbaI site, followed by a second copy of this enhancer attached to a homologous promoter, ending in a synthetic HindIII site. This vector DNA is cut with HindIII and the 6.3 kb linear plasmid is purified by agarose gel electrophoresis.
C) Vstavitev tPA/globin kombinacije v pSP62Pst33 (glej sl. 11) pSP62Pst33 (sl. 11) je plazmid, ki vsebuje 2,1 kbC) Insertion of the tPA/globin combination into pSP62Pst33 (see Fig. 11) pSP62Pst33 (Fig. 11) is a plasmid containing 2.1 kb
Pstl fragment DNA mišjega citomegalovirusa (MCMV), ki vključuje virusni, neposredni zgodnji (IE) promotor, vstavljen v Pstl mesto plazmida pSP62 (Boehringer Mannheira), kot je naznačeno na sliki. V Hindlll mesto pSP62Pst33 je vstavljen Hindlll fragment iz pD010. Izbran je plazmid pCGA26, v katerem je t-PA kodirno zaporedje vstavljeno tako, da se lahko prepisuje v smiselni orientaciji od MCMV IE promotorja.A Pstl fragment of murine cytomegalovirus (MCMV) DNA, which includes the viral immediate early (IE) promoter, was inserted into the Pstl site of plasmid pSP62 (Boehringer Mannheira) as indicated in the figure. The Hindlll fragment from pD010 was inserted into the Hindlll site of pSP62Pst33. Plasmid pCGA26 was selected in which the t-PA coding sequence was inserted such that it could be transcribed in the sense orientation of the MCMV IE promoter.
- 53 D) Vstavitev MCMV/tPA/globin enote v pFASV2911neo (glej sl.12)- 53 D) Insertion of the MCMV/tPA/globin unit into pFASV2911neo (see Fig. 12)
Plazmid pSV2911neo /F. Asselbergs et al., J, Mol. Biol. 189, 401-411 (1986)/ vsebuje neomicin (neo) fosfotransferazni gen iz transpozona TN5 v SV40 ekspresi jski kaseti (sl. 12). Tako podeli odpornost do neomicina in kanamicina, če je vpeljan v celice tkivne kulture sesalcev. pSV2911neo DNA pripravimo za kloniranje z rezanjem z BamHI, z obdelavo z govejo črevesno alkalno fosfatazo, dvema ekstrakcijama s fenolom, dvema s kloroformom, precipitacijo z alkoholom in končno jo raztopimo v TE. Plazmid pCGA26 režemo z restrikcijskim encimom Aecl, kateri reže zaporedje GT/ACAC na legi 345 v območju MCMV povečevalnik/promotor /K. Doersch-Haessler et al., Proc. Natl. Acad. Sci. USA 82, 8325-8329 (1985)/ in zaporedjePlasmid pSV2911neo /F. Asselbergs et al., J, Mol. Biol. 189, 401-411 (1986)/ contains the neomycin (neo) phosphotransferase gene from the TN5 transposon in an SV40 expression cassette (Fig. 12). It thus confers resistance to neomycin and kanamycin when introduced into mammalian tissue culture cells. pSV2911neo DNA was prepared for cloning by cutting with BamHI, treatment with bovine intestinal alkaline phosphatase, two phenol extractions, two chloroform extractions, alcohol precipitation, and finally dissolution in TE. Plasmid pCGA26 was cut with the restriction enzyme AecI, which cuts the sequence GT/ACAC at position 345 in the MCMV enhancer/promoter region /K. Doersch-Haessler et al., Proc. Natl. Acad. Sci. USA 82, 8325-8329 (1985)/ and sequence
GT/CGAC (lahko se reže tudi s Šali) za globinskim delom. Preko štrleče ostanke iz dveh baz, ki izhajajo po rezanju, zapolnimo z E.coli (velik fragment) DNA polimerazo I, sedaj tope konce povežemo z BamHI povezovalci (CGGATCCG) in te režemo z BamHI encimom. 3,8 kb fragment, ki nosi MCMV/tPA/globin enoto, sedaj 2 BamHI konci, čistimo preko agaroznega gela in nato vežemo na pSV2911neo DNA? pripravljeno, kot je opisano zgoraj, da dobimo ekspresijski plazmid pCGA28.GT/CGAC (can also be cut with Shali) after the deep part. The overhanging residues from the two bases that emerge after cutting are filled in with E.coli (large fragment) DNA polymerase I, now the blunt ends are ligated with BamHI linkers (CGGATCCG) and these are cut with BamHI enzyme. The 3.8 kb fragment carrying the MCMV/tPA/globin unit, now 2 BamHI ends, is purified through an agarose gel and then ligated to pSV2911neo DNA? prepared as described above to obtain the expression plasmid pCGA28.
E) Ekspresijski vektorji izpeljani iz pCGA28 pCGA42 je izpeljanka pCGA28, v kateri je neo kodirno zaporedje (med Bglll mestom in Sraal mestom) nadomeščeno s kodirnim zaporedjem higromicin odpornega gena. To dosežemo (glej sl. 13) z rezanjem plazmida pSV2911neo na njegovem edinem Smal mestu, vezanjem Bglll povezovalca (CAGATCTG) na DNA, sledi rezanje z Bglll. Izhajajoč veliki DNA fragment, ki sestoji iz vektorja minus neo kodirno zaporedje, čistimo na agaroznem gelu in vežemo na majhen BamHI fragment iz plazmida pLG89 /L. Gritz et al., Gene 25, 179-188 ( 19&3)/, enako čiščenega na agaroznem gelu, in ga vodimo v plazmida pCGA25c in pCGA25d, katera vsebujeta higromicin fosfotransferazni gen v smiselni oziroma nesmiselni orientaciji. Če se transficira v CHO DUKXB1 celice pri standardnih pogojih (glej primer 16), da pCGA25C 60 kolonij/^ig DNA odpornih na 0,2 ^ig/ml higromicina B, koncentracija, ki ubije CHO celice, ki vsebujejo plazmid ki ne kodira higromicin odpornosti, npr. pCGA28. V pCGA25c se zaporedja, ki kodirajo higromicin-B odpornost, nahajajo tako, da se v E.coli prepisujejo od Tn5 promotorja (kateri v transpozonu Tn5 prepiše kanamicin odporen gen). Tako 2,5 ml kulture Luria broth (LB), ki vsebuje 40 mg/1 higromicina-B, inokulirane z 0,05 ml, preko noči vzgojene, t.j. nasičene kulture E.coli DH1 bakterije (ki raste pod 50 mg/1 ampicilinsko selekcijo) doseže po 3 urah izpostavljanja zraku P^i 37°C najmanj 10-krat višjo gostoto bakterij, kot kadar testiramo bakterije s plazmidi, ki ne vsebujejo higromicinskega gena, delujočega v E.coli, kot pCGA25d, pCGA28 ali pAT153 /A.J. Twigg et al., Nature 283, 216-218 (1980)/. Funkcionalnost higromicin-B odpornega gena pri obeh, v celicah živalske tkivne kulture in v E.coli, močno olajša uporaba plazmida pCGA25c in njegovih izpeljank. Plazmid pCGA42 nato konstruiramo z vstavitvijoE) Expression vectors derived from pCGA28 pCGA42 is a derivative of pCGA28 in which the neo coding sequence (between the BglII site and the SraI site) has been replaced by the coding sequence of the hygromycin resistance gene. This is achieved (see Fig. 13) by cutting the plasmid pSV2911neo at its only SmaI site, ligating the BglII linker (CAGATCTG) to the DNA, followed by cutting with BglII. The resulting large DNA fragment, consisting of the vector minus the neo coding sequence, is purified on an agarose gel and ligated to a small BamHI fragment from plasmid pLG89 /L. Gritz et al., Gene 25, 179-188 (19&3)/, purified on an agarose gel, and introduced into plasmids pCGA25c and pCGA25d, which contain the hygromycin phosphotransferase gene in the sense and antisense orientations, respectively. When transfected into CHO DUKXB1 cells under standard conditions (see Example 16), pCGA25C yielded 60 colonies/µg DNA resistant to 0.2 µg/ml hygromycin B, a concentration that kills CHO cells containing a plasmid not encoding hygromycin resistance, e.g. pCGA28. In pCGA25c, the sequences encoding hygromycin-B resistance are located so that in E. coli they are transcribed from the Tn5 promoter (which in the Tn5 transposon transcribes the kanamycin resistance gene). Thus, a 2.5 ml Luria broth (LB) culture containing 40 mg/l hygromycin-B inoculated with 0.05 ml of an overnight, i.e. saturated culture of E.coli DH1 bacteria (growing under 50 mg/l ampicillin selection) reaches after 3 hours of exposure to air P^i 37°C at least 10 times higher bacterial density than when tested with plasmids that do not contain the hygromycin gene active in E.coli, such as pCGA25d, pCGA28 or pAT153 /A.J. Twigg et al., Nature 283, 216-218 (1980)/. The functionality of the hygromycin-B resistance gene in both animal tissue culture cells and in E.coli is greatly facilitated by the use of the plasmid pCGA25c and its derivatives. The plasmid pCGA42 is then constructed by inserting
BamHI fragmenta iz pCGA28, ki vsebuje MCMV/t-PA/beta-globin kaseto v pCGA25c. Njegova uporaba je, da prenesemo t-PA izražajoči gen v celice, ki jih ne moremo transformirati v geneticin odpornost ali katere so že geneticin odporne. Tudi pCGA42 je sposoben ekspresije svojega higromicin gena v E.coli, ki dopušča pCGA42, ki vsebuje E.coli DH1, da raste do gostot najmanj 10-krat večjih kot npr. pCGA28 vsebujoč E.coli, če testiramo kot je opisano zgoraj.BamHI fragment from pCGA28 containing the MCMV/t-PA/beta-globin cassette into pCGA25c. Its use is to transfer the t-PA expression gene into cells that cannot be transformed to geneticin resistance or that are already geneticin resistant. pCGA42 is also capable of expressing its hygromycin gene in E.coli, allowing pCGA42 containing E.coli DH1 to grow to densities at least 10-fold greater than, for example, pCGA28 containing E.coli, when tested as described above.
Plazmid pCGA28 vsebuje dve SacI mesti, eno v začetku povezovalca tik za MCMV promotorjem, drugo v t-PA cDNA. Zporedje med SacI mesti je izbrisano prvič z rezanjem z restrikcijskim encimom, čiščenjem večjega fragmenta preko agaroznega gela in cirkularizacijo te linearne DNA z uporaboPlasmid pCGA28 contains two SacI sites, one at the beginning of the linker just after the MCMV promoter, the other in the t-PA cDNA. The sequence between the SacI sites is deleted by first cutting with a restriction enzyme, purifying the larger fragment through an agarose gel, and circularizing this linear DNA using
DNA ligaze, ki tvori plazmid pCGA44 (glej sl. 12). Vsaka cDNA, klonirana v pravilni orientaciji v sedaj edinem SacI mestu pCGA44, učinkovito nadomesti t-PA kodirno zaporedje v pCGA28 in je učinkovito izražena.DNA ligase, which forms the plasmid pCGA44 (see Fig. 12). Each cDNA cloned in the correct orientation into the currently unique SacI site of pCGA44 effectively replaces the t-PA coding sequence in pCGA28 and is efficiently expressed.
pCGA42d je izpeljan iz pCGA42 z uničenjem 1,4 kb Sad fragmenta (glej sl. 13). V sedaj edinem Sad mestu cDNA lahko vstavimo drugo kot t-PA cDNA in se izraža na visokih nivojih v celicah tkivne kulture.pCGA42d is derived from pCGA42 by deletion of the 1.4 kb Sad fragment (see Fig. 13). In the currently unique Sad site, a cDNA other than t-PA cDNA can be inserted and is expressed at high levels in tissue culture cells.
PRIMER 5: Vstavitev cDNA , u-PA, t-PA in hibridnega PA v ekspresijski vektor pCGA28EXAMPLE 5: Insertion of cDNA, u-PA, t-PA and hybrid PA into the expression vector pCGA28
A) Vstavitev t-PA cDNA (glej sl. 15)A) Insertion of t-PA cDNA (see Fig. 15)
V tej konstrukciji vstavimo t-PA cDNA fragment iz plazmida ph.tPA4ScaI. Menimo, da je ta konstrukcija potrebna, da služi kot kontrola za vsako spremembo, ki bi se lahko nepazljivo pripetila med restrukturiranjem Seal mesta. 1,4 kb SacI fragment pridobimo iz plazmida po ph.tPA/jScal prebavi. Ekspresijski vektor pCGA28 tudi cepimo s SacI in 8,2 kb vektorski fragment izoliramo in defosforiliramo v 100 ^il reakcijske mešanice, ki vsebuje 0,1 mM Tris pH 8,0, 0,1 % SDS in 0,02 enoti bakterijske alkalne fosfataze. Sledeč inkubaciji pri 60°C 30 min., reakcijo ekstrahiramo dvakrat s fenolom in etrom in nato precipitiramo z etanolom. Usedlino raztopimo v vodi in njen alikvot uporabljamo za vezanje na 1,4 kb SacI fragment iz ph.tPA Seal. Mešanico za vezanje uporabljamo, da transformiramo E.coli HB101, in minilizirana DNA, pripravljena iz ampicilin-odpornih kolonij se prebavi s primernim restrikcij da ugotovimo, če je SacI vključek v želeni orientaciji. Plazmid, ki ima želeno orientacijo, se imenuje pBR1A. Plazmid s SacI fragmentom v nasprotni orientaciji je imenovan pBR1B.In this construct, we insert the t-PA cDNA fragment from the plasmid ph.tPA4ScaI. We believe that this construct is necessary to serve as a control for any changes that might inadvertently occur during the remodeling of the Seal site. The 1.4 kb SacI fragment is recovered from the plasmid after the ph.tPA/jScaI digestion. The expression vector pCGA28 is also cleaved with SacI and the 8.2 kb vector fragment is isolated and dephosphorylated in 100 µl of a reaction mixture containing 0.1 mM Tris pH 8.0, 0.1% SDS, and 0.02 units of bacterial alkaline phosphatase. Following incubation at 60°C for 30 min, the reaction is extracted twice with phenol and ether and then precipitated with ethanol. The precipitate is dissolved in water and an aliquot is used to bind to the 1.4 kb SacI fragment from ph.tPA Seal. The ligation mixture is used to transform E. coli HB101, and the minilized DNA prepared from ampicillin-resistant colonies is digested with the appropriate restriction enzyme to determine if the SacI insert is in the desired orientation. The plasmid with the desired orientation is called pBR1A. The plasmid with the SacI fragment in the opposite orientation is called pBR1B.
B) Vstavitev hibridne UPA^TPA^ cDNA (glej sl. 16)B) Insertion of hybrid UPA^TPA^ cDNA (see Fig. 16)
A BA B
V tej konstrukciji vstavimo hibridni UPA TPA cDNA fragment iz plazmida pUNC.tc v ekspresijski vektor pCGA28. pUNC.tc DNA se prebavi s Smal (primerjaj sl. 7), 1,24 kb fragment izoliramo in vežemo na SacI prebavljeno, defosforilirano 8,2 kb pCGA28 vektorsko DNA. E.coli HB101 celice transformiramo z mešanico za vezavo in kolonije, ki vsebujejo Sad vključek v želeni orientaciji, identificiramo s tem, da napravimo restrikcijske prebavke na minilizirani DNA. Plazmid s pUNC.tc DNA vključkom v želeni orientaciji je označen pBR2A in tisti z nasprotno orientacijo pBR2B.In this construction, the hybrid UPA TPA cDNA fragment from the plasmid pUNC.tc is inserted into the expression vector pCGA28. The pUNC.tc DNA is digested with SmaI (compare Fig. 7), the 1.24 kb fragment is isolated and ligated to SacI digested, dephosphorylated 8.2 kb pCGA28 vector DNA. E. coli HB101 cells are transformed with the ligation mixture and colonies containing the Sad insert in the desired orientation are identified by performing restriction digests on the minilized DNA. The plasmid with the pUNC.tc DNA insert in the desired orientation is designated pBR2A and that with the opposite orientation is designated pBR2B.
C) Vstavitev u-PA cDNA (glej sl. 17)C) Insertion of u-PA cDNA (see Fig. 17)
V zej konstrukciji vstavimo humano u-PA DNA v ekspresijski vektor pCGA28 in skupaj s pBR1 služi ta plazmid kot starševska plazmidna kontrola in potrjuje uporabnost vektorjev vrste pCGA28... Plazmid pcUK176 se prebavi s Smal, Ahalll (primerjaj sl. 4), 2,25 kb fragment izoliramo in vežemo na fosforiliran SacI povezovalec, kot je opisano zgoraj. Sledeč SacI prebavi, pridobimo 2,25 kb fragment in vežemo na SacI prebavljen, defosforiliran 8,2 kb pCGA28 DNA fragment. E.coli HB101 se transformira in kolonije, ki varujejo želeni plazmid, identificiramo s prebavo minilizirane DNA z restrikcijskimi encimi. Plazmid s humano u-PA DNA v pravilni orientaciji je označen pBR23A in oni v nasprotni orientaciji pBR3B.In this construction, human u-PA DNA is inserted into the expression vector pCGA28 and together with pBR1, this plasmid serves as a parental plasmid control and confirms the utility of vectors of the pCGA28 type... Plasmid pcUK176 is digested with SmaI, AhalII (compare Fig. 4), the 2.25 kb fragment is isolated and ligated to the phosphorylated SacI linker as described above. Following SacI digestion, the 2.25 kb fragment is obtained and ligated to the SacI digested, dephosphorylated 8.2 kb pCGA28 DNA fragment. E.coli HB101 is transformed and colonies harboring the desired plasmid are identified by digestion of the minilized DNA with restriction enzymes. The plasmid with human u-PA DNA in the correct orientation is designated pBR23A and those in the opposite orientation pBR3B.
D) Vstavitev hibridne TPA^UPA^. cDNA (sl. 18)D) Insertion of hybrid TPA^UPA^. cDNA (Fig. 18)
A BA B
Tukaj vstavimo hibridno TPA UPA cDNA iz plazmida ptNC.UC v ekspresijski vektor pCGA28. 2,75 kb Smal (prisoten v vektorju), Ahalll fragment izoliramo iz ptNC.UC DNA, vežemo na fosforiliran SacI povezovalec, povezovalec veže 2,75 kb fragment, pridobljen in vezan na SacI prebavljen, defosforiliran vektor DNA in želene kolonije identificiramo, kot je opisano zgoraj. Plazmid s ptNC.UC DNA vključkom v pravilni orientaciji imenujemo pBR4A.Here, we insert the hybrid TPA UPA cDNA from the plasmid ptNC.UC into the expression vector pCGA28. The 2.75 kb SmaI (present in the vector), AhalII fragment is isolated from the ptNC.UC DNA, ligated to a phosphorylated SacI linker, the linker binds the 2.75 kb fragment obtained and ligated to the SacI digested, dephosphorylated vector DNA, and the desired colonies are identified as described above. The plasmid with the ptNC.UC DNA insert in the correct orientation is called pBR4A.
PRIMER 6: Konstrukcija ekspresijskega vektorja kvasa, ki vsebuje pHO5 promotor, invertazno signalno zaporedje in t-PA kodirno območjeEXAMPLE 6: Construction of a yeast expression vector containing a pHO5 promoter, an invertase signal sequence and a t-PA coding region
A) Sinteza oligodeoksiribonukleotidov za invertazno signalno zaporedje:A) Synthesis of oligodeoxyribonucleotides for the invertase signal sequence:
Št iri oligodeoksiribonukleotide: 1-1, 1-2, 1-3, 1-4 sintetiziramo z DNA sintetizatorjem (model 3θΟΒ Applied Biosystems). Po deblokiranju sintetične fragmente čistimo na 12 % poliakrilamidnem gelu, ki vsebuje 8 M sečnino. Brez soli, čiste oligodeoksiribonukleotide dobimo z uporabo Sep. Pak (Waters Associates). Ti fragmenti predstavljajo dvojni heliks, kateri kodira invertazno signalno zaporedje s pogosto uporabljenimi kodoni kvasa.Four oligodeoxyribonucleotides: 1-1, 1-2, 1-3, 1-4 were synthesized using a DNA synthesizer (model 3θΟΒ Applied Biosystems). After deblocking, the synthetic fragments were purified on a 12% polyacrylamide gel containing 8 M urea. Salt-free, pure oligodeoxyribonucleotides were obtained using Sep. Pak (Waters Associates). These fragments represent a double helix encoding the invertase signal sequence with commonly used yeast codons.
HindlllHindllll
EcoRI MetLeuLeuGlnAlaPheLeuPheLeuLeuEcoRI MetLeuLeuGlnAlaPheLeuPheLeuLeu
1-1 5'AATTCATGCTTTTGCAAGCTTTCCTTTTCCTTTT 3'1-1 5'AATTCATGCTTTTGCAAGCTTTCCTTTTCCTTTT 3'
1-2 3' GTACGAAAACGTTCGAAAGGAAAAGGAAAACCGAC 5'1-2 3' GTACGAAAACGTTCGAAAGGAAAAGGAAAACCGAC 5'
AlaGlyPheAlaAlaLysIleSerAlaAlaGlyPheAlaAlaLysIleSerAla
1-3 5*GGCTGGTTTTGCAGCCAAAATATCTGCATCTTAGCGTC 3'1-3 5*GGCTGGTTTTGCAGCCAAATATCTGCATCTTAGCGTC 3'
1-4 3’CAAAACGTCGGTTTTATAGACGTAGAATCGCAGAGCT 5'1-4 3'CAAAACGTCGGTTTTATAGACGTAGAATCGCAGAGCT 5'
Hgal_Hgal_
XholXhol
B) Subkloniranje invertaznega signalnega zaporedja v plazmidu p31B) Subcloning of the invertase signal sequence in the p31 plasmid
a) Priprava vektorja:a) Vector preparation:
1,5 P31R/SS-TPA 2 (evropska patentna prijava št. 143,081 ) se prebavlja z 10 U EcoRI (Boehringer) v 50 10 mM1.5 P31R/SS-TPA 2 (European Patent Application No. 143,081 ) is digested with 10 U EcoRI (Boehringer) in 50 10 mM
Tris.HCl pH 7,5, 6 mM MgC^, 100 mM NaCl, 6 mM merkaptoetanola “;· :-:59 .eno uro pri 37°C. Po dodatku 1 2,5 M NaCl dodamo 10 U Xhol (Boehringer) in inkubiramo pri 37°C eno uro. 4,2 kb vektor izoliramo na 0,8 % preparativnem agaroznem gelu. Rezino gela prenesemo na Micro Colloidor cev (Sartorius GmbH), prekrito z 200 )il TE in elektroeluiramo (z elektroforezo pri 90 mA 50 min.). TE raztopino zberemo in precipitiramo v 2,5 volumnih absolutnega etanola po dodatku 0,1 volumna 10 χ TNE. DNA usedlino speremo z mrzlim 80 % etanolom in posušimo v vakuumu. DNA resuspendiramo v 6 ^il TE (40 pmolov/^l).Tris.HCl pH 7.5, 6 mM MgCl^, 100 mM NaCl, 6 mM mercaptoethanol “;· :-:59 .one hour at 37°C. After the addition of 1 2.5 M NaCl, 10 U Xhol (Boehringer) was added and incubated at 37°C for one hour. The 4.2 kb vector was isolated on a 0.8% preparative agarose gel. The gel slice was transferred to a Micro Colloidor tube (Sartorius GmbH), covered with 200 )il TE and electroeluted (by electrophoresis at 90 mA for 50 min.). The TE solution was collected and precipitated in 2.5 volumes of absolute ethanol after the addition of 0.1 volume of 10 χ TNE. The DNA pellet was washed with cold 80% ethanol and dried in vacuo. The DNA was resuspended in 6 ^il TE (40 pmol/^l).
b) Renaturlranje (annealing) oligodeoksiribonukleotidov (1-1, 1-2, 1-3, 1--4), kinacija in vezava z vektorjemb) Annealing of oligodeoxyribonucleotides (1-1, 1-2, 1-3, 1--4), kinination and ligation with the vector
Raztopino, ki vsebuje 10 pmolov vsakega od štirih deoksiribonukleotidov v 10 jil 0,5 M Tris.HCI pH8, inkubiramo pri 95°C 5 minut na vodni kopeli. Vodno kopel počasi hladimo do 30°C v razdobju 5 ur. K tej renaturacijski mešanici dodamo 2 pl vsakega od 0,1 M MgCl2, 0,1 M NaCl, 30 mM DTT, 4 mM ATP in 8 U (1 2*1) polinukleotid kinaze (Boehringer). Kinacija poteka pri 37°C eno uro. Renaturirane, kinazirane oligodeoksiribonukleotide in 60 pmolov p31R/SS-TPAA2 rezanega vektorja (1,5 ^il) vežemo s 400 U (1 ^il) T4 DNA ligaze (Biolabs) pri 14°C 17 ur. Reakcijo ustavimo z inkubacijo pri 65°C 10 minut. 10 pl te vezalne mešanice uporabljamo za transformiranje E.coli HB101 Ca++ celic /M. Dagert and S.D. Ehrlich, Gene 56, pA solution containing 10 pmoles of each of the four deoxyribonucleotides in 10 µl of 0.5 M Tris.HCl pH8 was incubated at 95°C for 5 minutes in a water bath. The water bath was slowly cooled to 30°C over a period of 5 hours. To this renaturation mixture were added 2 µl each of 0.1 MMgCl2 , 0.1 M NaCl, 30 mM DTT, 4 mM ATP and 8 U (1 2*1) of polynucleotide kinase (Boehringer). Kination was carried out at 37°C for one hour. The renatured, kinased oligodeoxyribonucleotides and 60 pmoles of p31R/SS-TPAA2 cut vector (1.5 µl) were ligated with 400 U (1 µl) of T4 DNA ligase (Biolabs) at 14°C for 17 hours. The reaction was stopped by incubation at 65°C for 10 minutes. 10 µl of this ligation mixture is used to transform E.coli HB101 Ca++ cells /M. Dagert and SD Ehrlich, Gene 56, p
23-28 (1979)/. Izberemo 20 amp kolonij. DNA pripravimo s hitrim izolacijskim postopkom (D.S. Holmes and M. Quigley. Anal. Biochem. 114, 193-197 (1981)/. DNA se prebavi z EcoRI in Xhol, radiomarkirano pri EcoRI koncu in analiziramo na 6 %23-28 (1979)/. 20 amp colonies are selected. DNA is prepared by the rapid isolation procedure (D.S. Holmes and M. Quigley. Anal. Biochem. 114, 193-197 (1981)/. DNA is digested with EcoRI and XhoI, radiolabeled at the EcoRI end, and analyzed for 6%
66' poliakrilamidnem gelu, ki vsebuje 8 M sečnino, z uporabo radiomarkiranega pBR322 Haelll reza DNA, kot označevalca. Opazimo pasove pravilne velikosti za DNA, dobljeno iz vseh 20 klonov. En klon raste v 100 ml LS medija, ki vsebuje 100 ^ig/ml ampicilina. Plazmid DNA izoliramo in ga omenjamo kot p31RIT-12.66' polyacrylamide gel containing 8 M urea, using radiolabeled pBR322 Haelll DNA cut as a marker. Bands of the correct size are observed for DNA obtained from all 20 clones. One clone grows in 100 ml of LS medium containing 100 µg/ml ampicillin. The plasmid DNA is isolated and referred to as p31RIT-12.
C) Konstrukcija pJDB207/PH05-I-TPA (glej sl. 19)C) Construction of pJDB207/PH05-I-TPA (see Fig. 19)
a) Priprava vektorja:a) Vector preparation:
Tri ^ig pJDB207/PH05-TPA18 (evropska patentna prijava št. 143,081) inkubiramo pri 37°C eno uro z 10 U BamHI v 50 10 mM Tris.HCl pH 7,5, 6 mM MgCl9, 100 mM NaCl, 6 mM merkaptoetanola. Alikvot pregledamo na 1 % agaroznem gelu v TBE pufru, da potrdimo popolno prebavo. Prebavek inkubiramo pri 65°C 10 min. Nato dodamo 0,5 ^il 5 M NaCl, sledi 15 U Xhol (Boehringer) . To inkubiramo pri 37°C eno uro. 6,8 kb vektor izoliramo na 0,8 % preparativnem agaroznem gelu. DNA ekstrahiramo z elektroeluiranjem in po precipitaciji raztopimo v TE.Three µg of pJDB207/PH05-TPA18 (European Patent Application No. 143,081) were incubated at 37°C for one hour with 10 U BamHI in 50 mM Tris.HCl pH 7.5, 6 mM MgCl9 , 100 mM NaCl, 6 mM mercaptoethanol. An aliquot was examined on a 1% agarose gel in TBE buffer to confirm complete digestion. The digest was incubated at 65°C for 10 min. Then 0.5 µl of 5 M NaCl was added, followed by 15 U Xhol (Boehringer). This was incubated at 37°C for one hour. The 6.8 kb vector was isolated on a 0.8% preparative agarose gel. The DNA was extracted by electroelution and, after precipitation, dissolved in TE.
b) Xhol prebavek p31/PHO5-TPA18;b) XhoI digest of p31/PHO5-TPA18;
yig p31/PHO5-TPA 18 (evrop.pat.prijava št.143,081) inkubiramo pri 37°C eno uro s 60 U Xhol (15 U/j41) v 200 1 10 mM Tris.HCl pH 8, 6 mMyig p31/PHO5-TPA 18 (European Patent Application No. 143,081) was incubated at 37°C for one hour with 60 U Xhol (15 U/ml) in 200 l 10 mM Tris.HCl pH 8.6 mM
MgClg, 150 mM NaCl, 6 mM merkaptoetanola, ekstrahiramo z enakim volumnom fenolkloroforma in precipitiramo v etanolu.MgClg, 150 mM NaCl, 6 mM mercaptoethanol, extracted with an equal volume of phenolchloroform and precipitated in ethanol.
c) Delni Pstl prebavek Xhol reza p31/PHO5-TPAl8c) Partial Pstl digestion of XhoI cut p31/PHO5-TPAl8
Precipitirani Xhol rez P31/PHO5-TPA18 DNA resuspendiramo v 250 ^il 10 mM Tris.HCl pH 7,5, 6 mM MgCl2, 50 mM NaCl, 6 mM merkaptoetanola, 2,5 mg homidijevega bromida, inkubiramo pri 37°C 35 minut z 22,5 U Pstl in ekstrahiramo z enakim volumnom fenola in zatem z enakim volumnom kloroforma-izoamilalkohola (50:1). 1,6 kb fragment izoliramo na 1 % preparativ61 nem agaroznem gelu. DNA ekstrahiramo z elektroeluiranjem in precipitiramo /vključek 1/.The precipitated XhoI cut P31/PHO5-TPA18 DNA was resuspended in 250 µl of 10 mM Tris.HCl pH 7.5, 6 mMMgCl2 , 50 mM NaCl, 6 mM mercaptoethanol, 2.5 mg chromidium bromide, incubated at 37°C for 35 minutes with 22.5 U PstI and extracted with an equal volume of phenol and then with an equal volume of chloroform-isoamyl alcohol (50:1). The 1.6 kb fragment was isolated on a 1% preparative agarose gel. The DNA was extracted by electroelution and precipitated (entry 1).
d) Sall-Xhol prebavek p31RIT-12:d) Sall-Xhol digestion of p31RIT-12:
Trideset ^ig p31RIT-12 inkubiramo pri 37°C eno uro s 60 U Šali (Boehringer 12 U/^il) in 60 U Xhol (15 U/jil) v 200 p: 10 mM Tris.HCl pH 8, 6 mM MgCl2, 150 mM NaCl, 6 mM merkaptoeta nola, ekstrahiramo z enakim volumnom fenol^-kloroforma in precipitiramo v etanolu. 869 bp fragment izoliramo na 1,2 % preparativnem agaroznem gelu. DNA ekstrahiramo z elektroeluira njem, razsoljenim preko DE-52, in precipitiramo v etanolu.Thirty µg of p31RIT-12 was incubated at 37°C for one hour with 60 U of SalI (Boehringer 12 U/µl) and 60 U of Xhol (15 U/µl) in 200 µl of 10 mM Tris.HCl pH 8, 6 mMMgCl2 , 150 mM NaCl, 6 mM mercaptoethanol, extracted with an equal volume of phenol-chloroform and precipitated in ethanol. The 869 bp fragment was isolated on a 1.2% preparative agarose gel. The DNA was extracted by electroelution, desalted over DE-52, and precipitated in ethanol.
e) Hgal prebavek Sall-Xhol reza p3lRIT-12e) Hgal digestion of Sall-XhoI cut p3lRIT-12
Sall-Xhol rez p31RIT-12 resuspendiramo v 100 pl 6 mM Tris.HCl pH 7,5, 10 mM MgCl2, 50 mM NaCl, 1 mM ditiotreitola, 10 mg govejega serum albumina in inkubiramo pri 37°C eno uro s 6 U Hgal (Biolabs, 0,5 U /p 1). 600 bp fragment izoliramo na 1,2% agaroznem gelu. DNA ekstrahiramo z elektroeluiranjem in precipitiramo v etanolu.The Sal-XhoI cut of p31RIT-12 was resuspended in 100 µl of 6 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 50 mM NaCl, 1 mM dithiothreitol, 10 mg bovine serum albumin and incubated at 37°C for one hour with 6 U Hgal (Biolabs, 0.5 U/µl). The 600 bp fragment was isolated on a 1.2% agarose gel. The DNA was extracted by electroelution and precipitated in ethanol.
f) Renaturiranje povezovalnih oligonukleotidov pmolov dveh oligodeoksiribonukleotidov, ki imataf) Renaturation of linker oligonucleotides pmoles of two oligodeoxyribonucleotides having
Hgal PstlHgal Pstl
CTGCATCTTACCAAGTGATCTGCA 3'CTGCATCTTACCAAGTGATCTGCA 3'
3’AGAATGGTTCACTAG 5* v 10 0,5 mM Tris.HCl pH 8 v silikonizirani epruveti. Raztopino inkubiramo pri 95°C 5 min. in nato počasi ohladimo na sobno temperaturo čez noč.3’AGAATGGTTCACTAG 5* in 10 0.5 mM Tris.HCl pH 8 in a siliconized tube. The solution is incubated at 95°C for 5 min. and then slowly cooled to room temperature overnight.
zaporedjesequence
5' suspendiramo5' suspend
EppendorfoviEppendorfs
g) Kinacija povezovalcag) Kination of the linker
H gornji raztopini dodamo 2 yl 0,1 M KC1, 2 ^il 0,1 M MgCl2, 3 )il 30 mM DTT, 1 ^1 200 mM ATP, δ U polinukleotida (8 U/^il). To inkubiramo pri 37°C eno uro.To the above solution, add 2 µl of 0.1 M KC1, 2 µl of 0.1 M MgCl2 , 3 µl of 30 mM DTT, 1 µl of 200 mM ATP, δ U polynucleotide (8 U/µl). This was incubated at 37°C for one hour.
h) Vezava Hgal fragmenta iz p31RIT-12 s kinaziranim povezovalcemh) Binding of the Hgal fragment from p31RIT-12 with a kinased linker
Raztopino kinaziranega povezovalca prenesemo v epruvetko, ki vsebuje suh Hgal fragment, in dodamo 400 U DNA ligaze. Raztopino inkubiramo pri sobni temperaturi (21 do 22°C) 90 minut, razredčimo na 100 )il s TE in ekstrahiramo z enakim volumnom fenola/kloroforma. Fragment precipitiramo z dodajanjem 0,6 volumna izopropanola in 0,1 volumna 3 M natrijevega acetata pri sobni temperaturi k vodni raztopini.The kinased linker solution was transferred to a tube containing the dry Hgal fragment and 400 U of DNA ligase was added. The solution was incubated at room temperature (21 to 22°C) for 90 minutes, diluted to 100 µl with TE, and extracted with an equal volume of phenol/chloroform. The fragment was precipitated by adding 0.6 volumes of isopropanol and 0.1 volumes of 3 M sodium acetate at room temperature to the aqueous solution.
i) BamHI-Pstl prebavek gornjegai) BamHI-Pstl digest of the upper
Gornja suha DNA se prebavlja v 10 U BamHI in 10 U Pstl v 20 ^μΐ 10 mM Tris.HCl phT 7,5, 100 mM MgCl2, 6 mM merkaptoetanola eno uro pri 37°C. Po razredčenju na 100 ^il raztopino ekstrahiramo z enakim volumnom fenola/kloroforma, in vodno plast precipitiramo v izopropanolu /vključek 2/.The above dried DNA is digested in 10 U BamHI and 10 U Pstl in 20 µl of 10 mM Tris.HClpH 7.5, 100 mM MgCl2 , 6 mM mercaptoethanol for one hour at 37°C. After dilution to 100 µl, the solution is extracted with an equal volume of phenol/chloroform, and the aqueous layer is precipitated in isopropanol /entry 2/.
j) Vezanje treh fragmentovj) Binding of three fragments
100 fmolov pJDB207/PH05-TPA1S BamHI-XhoI reza vektorskega fragmenta, 200 fmolov vsakega od drugih dveh vstavljenih fragmentov /1 in 2/ vežemo v 10 50 mM Tris-HCl pH 7,5, mM MgCl2, 10 mM DTT, 2 mM ATP, 0,5 )ig želatine s 400 U T^100 fmoles of the pJDB207/PH05-TPA1S BamHI-XhoI cut vector fragment, 200 fmoles of each of the other two inserted fragments /1 and 2/ were ligated in 10 50 mM Tris-HCl pH 7.5, mM MgCl2 , 10 mM DTT, 2 mM ATP, 0.5 )ig gelatin with 400 UT^
- 63 DNA ligaze 16 ur pri 15°C. Reakcijo ustavimo z inkubiranjem pri 65°C 10 min. 5 ^1 te vezalne mešanice uporabljamo za transformiranje celic E.coli HB101 CA . 10 amp* kolonij poberemo in DNA pripravimo po hitrem izolacijskem postopku. Pri analizi z EcoRI, Pstl in BamHI-Kindlll opazimo fragmente pravilne velikosti. En klon raste v 100 ml LB medija, ki vsebuje 100 ^jg/ml ampicilina. Plazmid DNA izoliramo in ga omenjamo kot pJDB 207/PH05-I-TPA.- 63 DNA ligase for 16 hours at 15°C. The reaction is stopped by incubation at 65°C for 10 min. 5 µl of this ligation mixture is used to transform E.coli HB101 CA cells. 10 amp* colonies are picked and DNA is prepared by a rapid isolation procedure. Analysis with EcoRI, PstI and BamHI-KindlI shows fragments of the correct size. One clone grows in 100 ml of LB medium containing 100 µg/ml ampicillin. The plasmid DNA is isolated and referred to as pJDB 207/PH05-I-TPA.
PRIMER 7: Konstrukcija plazmida pCSl6/UPA, ki obsega u-PA kodirno območjeEXAMPLE 7: Construction of plasmid pCS16/UPA comprising the u-PA coding region
A) Konstrukcija plazmida pCSl6 (glej sl. 20)A) Construction of plasmid pCSl6 (see Fig. 20)
1,5 kb Pstl-BamHI fragment plazmida pUN121 /B.Nilsson et al., Nucl. Acids Res. 11 . 8019-8030 (1983)/, ki obsega cl gen faga lambda in del tetraciklin odpornega gena, kloniramo v pUCl8 /J. Norrander et al., Gene 26, 101-106 ( 1983)/, režemo s Pstl in BamHI. Izhajajoči klon se prebavi s Pstl. 3’preko viseče konce odstranimo v reakciji s DNA polimerazo inA 1.5 kb Pstl-BamHI fragment of plasmid pUN121 /B.Nilsson et al., Nucl. Acids Res. 11 . 8019-8030 (1983)/, which comprises the cl gene of phage lambda and part of the tetracycline resistance gene, is cloned into pUCl8 /J. Norrander et al., Gene 26, 101-106 (1983)/, cut with Pstl and BamHI. The resulting clone is digested with Pstl. The 3' overhanging ends are removed in a reaction with DNA polymerase and
Xhol povezovalce vežemo na tope konce. Po prebavi s Xhol molekulo z vezavo ponovno spremenimo v krožno obliko. Alikvot vezalne mešanice uporabljamo, da transformiramo Ca++ obdelane E.coli HB101 celice. Analiziramo DNA posameznih, ampicilin odpornih, transformiranih kolonij. Enega od več pravilnih klonov izberemo in ga omenjamo kot pCSl6.XhoI linkers are ligated to the blunt ends. After digestion with XhoI, the molecule is recircularized by ligation. An aliquot of the ligation mixture is used to transform Ca++ -treated E.coli HB101 cells. The DNA of individual ampicillin-resistant transformed colonies is analyzed. One of several correct clones is selected and referred to as pCSl6.
B) Konstrukcija plazmida pCSl6/UPA (glej sl. 21)B) Construction of plasmid pCSl6/UPA (see Fig. 21)
Urokinazno cDNA, kot je vsebovana v plazmidu pcUK176 (glej primer 2), subkloniramo v plazmidu pCSl6. Subklonirana cDNA sega od Smal mesta v 5’neprevajanem območju (sl. 4) do PvuII mesta pri legah nukleotidov 1439-1444 v 3’neprevajanem območju (oštevilčenje v smislu sl. 3).The urokinase cDNA, as contained in the plasmid pcUK176 (see Example 2), was subcloned into the plasmid pCS16. The subcloned cDNA extends from the SmaI site in the 5'untranslated region (Fig. 4) to the PvuII site at nucleotide positions 1439-1444 in the 3'untranslated region (numbering as in Fig. 3).
^ig plazmida pcUK176 se prebavi s PvuII. 379 bp PvuII fragment izoliramo od drugih fragmentov na 1,5 % agaroznem gelu v Tris-boratnem EDTA pufru pH 8,3. DNA elektroeluiramo, čistimo z DE52 (Whatman) ionsko izmenjalno kromatografijo in precipitiramo z etanolom. 1,2 ^g eno vlaknatih Xhol povezovalcev (5’-CCTCGAGG-3?) fosforiliramo na 5’koncih, segrevamo 10 min. pri 75°C, med hlajenjem na sobno temperaturo se sami renaturirajo in jih shranimo pri -20°C. 0,9 )ig kinaznih, dvojno vlaknatih Xhol povezovalcev^ vežemo pri 80-kratnem molarnem prebitku na tope konce 379 bp PvuII fragmenta pcUK176 (glej^ig of plasmid pcUK176 is digested with PvuII. The 379 bp PvuII fragment is isolated from other fragments on a 1.5% agarose gel in Tris-borate EDTA buffer pH 8.3. The DNA is electroeluted, purified by DE52 (Whatman) ion exchange chromatography and precipitated with ethanol. 1.2 ^g of single-stranded XhoI linkers (5'-CCTCGAGG-3? ) are phosphorylated at the 5' ends, heated for 10 min. at 75°C, self-renatured during cooling to room temperature and stored at -20°C. 0.9 )ig of kinased, double-stranded XhoI linkers^ are ligated in 80-fold molar excess to the blunt ends of the 379 bp PvuII fragment of pcUK176 (see
povezovalnih molekul odstranimo s precipitacijo z 0,54 volumni izopropanola v prisotnosti 10 mM EDTA in 300 mM natrijevega acetata pH 6,0, 30 minut pri sobni temperaturi. DNA se prebavi s Xhol in BamHI. 121 bp BamHI-XhoI fragment izoliramo na 1,5% agaroznem gelu v Tris-borat-EDTA pufru pH 8,3. 6 plazmida pcUK176 se prebavi s Smal in BamHI. 1,3 kb Smal-BamHI fragment, ki obsega večino u-PA kodirnega zapored ja, izoliramo. 6 ^ig plazmida pCSl6 se prebavi s Smal in Xhol. 2,7 kb vektorski fragment izoliramo. DNA fragmente elektroeluiramo iz gela in precipitiramo z etanolom. 0,2 pmolov 1,3 kb Smal-BamHI fragmenta, 0,2 pmolov 121 bp BamHI-XhoI fragmenta (oba fragmenta skupaj obsegata celotno u-PA kodirno zaporedje) in 0,1 praolThe linker molecules are removed by precipitation with 0.54 volumes of isopropanol in the presence of 10 mM EDTA and 300 mM sodium acetate pH 6.0 for 30 minutes at room temperature. The DNA is digested with XhoI and BamHI. The 121 bp BamHI-XhoI fragment is isolated on a 1.5% agarose gel in Tris-borate-EDTA buffer pH 8.3. 6 µg of plasmid pcUK176 is digested with SmaI and BamHI. A 1.3 kb SmaI-BamHI fragment, comprising most of the u-PA coding sequence, is isolated. 6 µg of plasmid pCS16 is digested with SmaI and XhoI. A 2.7 kb vector fragment is isolated. The DNA fragments are electroeluted from the gel and ethanol precipitated. 0.2 pmol of 1.3 kb SmaI-BamHI fragment, 0.2 pmol of 121 bp BamHI-XhoI fragment (both fragments together comprise the entire u-PA coding sequence) and 0.1 pmol of
2,7 kb vektorskega fragmenta, vežemo v 10 jil 60 mM Tris.HCI pH 7,5, 10 mM MgCl2, 5 mM DTT, 3,5 mM ATP in 400 enotah DNA ligaze pri 15°C. Eno in3 jjl-ske alikvote vezalne mešanice dodamo k 100 ^il Ca++ tretiranih E.coli HB101 celic. Transformiranje poteka, kot je opisano /A. Hinnen et. al., Proc.Natl. Acad. Sci. USA 75, 1929 (1978)/. 12 ampicilin odpornih kolonij raste v LB mediju, ki vsebuje 100 mg/1 ampicilina. DNA izolira mo v smislu Holmes et al. /Anal. Biochem. 114, 193 (1981)/ in analiziramo po EcoRI, PvuII in Xhol restrikcijskih prebavah. En klon s pričakovanimi restrikcijskimi fragmenti omenjamo kot PCS16/UPA.The 2.7 kb vector fragment was ligated in 10 µl of 60 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 5 mM DTT, 3.5 mM ATP and 400 units of DNA ligase at 15°C. One and a third µl aliquots of the ligation mixture were added to 100 µl of Ca++ treated E.coli HB101 cells. Transformation was carried out as described /A. Hinnen et. al., Proc.Natl. Acad. Sci. USA 75, 1929 (1978)/. 12 ampicillin resistant colonies were grown in LB medium containing 100 mg/l ampicillin. DNA was isolated according to Holmes et al. /Anal. Biochem. 114, 193 (1981)/ and analyzed by EcoRI, PvuII and XhoI restriction digestions. One clone with the expected restriction fragments is referred to as PCS16/UPA.
PRIMER 8: Konstrukcija plazmida pJDB207/PH05-I-UPA (sl. 22) pJD3207/PHQ5-I-UPA vsebuje PHO5 promotor, invertazno signalno zaporedje, kodirno zaporedje zrele urokinaze in PHO5 prepisovalni terminator v vrsti, razporejeno eno za drugimi, klonirano v pJDB207 ekspresijskem vektorju kvasa.EXAMPLE 8: Construction of plasmid pJDB207/PH05-I-UPA (Fig. 22) pJD3207/PHQ5-I-UPA contains the PHO5 promoter, invertase signal sequence, mature urokinase coding sequence and PHO5 transcription terminator in a row, arranged one after the other, cloned in the pJDB207 yeast expression vector.
^ig plazmida pCSl6/UPA se prebavi do konca s 40 enotami EcoRI. Po fenolni ekstrakciji in etanolni precipitaciji z EcoRI prebavljeno DNA nadalje režemo s Taql pri 65°C. Izhajajoče fragmente ločimo na preparativnem 1,2 % agaroznem gelu. 462 bp TaqI-EeoRI fragment izoliramo z elektroeluiranjem iz gela in precipitiramo z etanolom.The plasmid pCS16/UPA is digested to completion with 40 units of EcoRI. After phenol extraction and ethanol precipitation with EcoRI, the digested DNA is further digested with TaqI at 65°C. The resulting fragments are separated on a preparative 1.2% agarose gel. The 462 bp TaqI-EeoRI fragment is isolated by electroelution from the gel and ethanol precipitated.
Oligodezoksiribonukleotidni povezovalec s formulo (I) 5'-CTGCAAGCAATGAACTTCATCAAGTTCCAT-3’ (II) 3’- TCGTTACTTGAAGTAGTTCAAGGTAGC-5’ vežemo na Taci mesto DNA fragmenta. Povezovalec obnovi 5'konec kodirnega zaporedja zrelega u-PA (nukleotidi 130-154, sl. 3) in uvede zlitje v okviru z invertaznim signalnim zaporedjem. 5’-CTGCA zaporedje povezovalca zapolni odgovarjajoči 3’poglobijeni del invertaznega signalnega zaporedja, ustvarjenega s Hgai cepitvijo.An oligodeoxyribonucleotide linker of the formula (I) 5'-CTGCAAGCAATGAACTTCATCAAGTTCCAT-3' (II) 3'- TCGTTACTTGAAGTAGTTCAAGGTAGC-5' is ligated to the Taci site of the DNA fragment. The linker restores the 5' end of the mature u-PA coding sequence (nucleotides 130-154, Fig. 3) and introduces an in-frame fusion with the invertase signal sequence. The 5'-CTGCA sequence of the linker fills in the corresponding 3' recessed portion of the invertase signal sequence generated by Hgai cleavage.
300 pmolov vsakega od oligodezoksinukleotidov I in II fosforiliramo in renaturiramo. 5,25 ^ig (600 pmolov) fosforilirane dvojno-vlaknate povezovalne DNA vežemo na 1,7 (5,6 pmolov) 462 bp Taqi-EcoRI fragmenta (glej zgoraj) v 175 y 60 mM Tris-HCl pH 7,5, 10 mM MgCl2, 1 mM ATP, 5 mM DTT in 800 enotah T4 DNA ligaze pri 15°C 16 ur. T4 DNA ligazo inaktiviramo 10 min. pri 85°C. Prebitek povezovalcev odstranimo s precipitiranjem v prisotnosti 10 mM EDTA 300 mM natrijevega acetata pH 6,0 in 0,54 volumna izopropanola. DNA se prebavi s Pstl. Edini 312 bp fragment, ki vsebuje povezovalec, pritrjen na DNA zaporedja, ki kodirajo u-PA do nukleotida 436 (Pstl mesto, glej sl. 3) izoliramo. DNA fragment čistimo z elektroeluiranjem in precipitiramo z etanolom.300 pmoles of each of oligodeoxynucleotides I and II were phosphorylated and renatured. 5.25 µg (600 pmoles) of phosphorylated double-stranded linker DNA was ligated to 1.7 (5.6 pmoles) of the 462 bp Taqi-EcoRI fragment (see above) in 175 µl of 60 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 1 mM ATP, 5 mM DTT and 800 units of T4 DNA ligase at 15°C for 16 hours. T4 DNA ligase was inactivated for 10 min. at 85°C. Excess linkers were removed by precipitation in the presence of 10 mM EDTA, 300 mM sodium acetate pH 6.0 and 0.54 volumes of isopropanol. The DNA was digested with PstI. A single 312 bp fragment containing a linker attached to the DNA sequences encoding u-PA up to nucleotide 436 (PstI site, see Fig. 3) was isolated. The DNA fragment was purified by electroelution and ethanol precipitated.
Plazmid pCSl6/UPA se prebavi s Xhol in Pstl. 1007 bp Pstl-Xhol fragment izoliramo in čistimo. Ta fragment vsebuje večino kodirnega zaporedja za urokinazo.Plasmid pCS16/UPA is digested with XhoI and Pstl. A 1007 bp Pstl-XhoI fragment is isolated and purified. This fragment contains most of the coding sequence for urokinase.
Plazmid p31RIT-12 (glej primer 6b) se prebavi s Šali in Xhoi. 382 bp SalI~XhoI fragment izoliramo iz gela z elektro eluiranjem in etanolno precipitacijo. Fragment se nadalje prebavi z BamHI in Hgai. 591 bp BamHI-Hgal fragment, kateri vsebuje PH05 promotorsko območje in invertazno signalno zaporedje, izoliramo.Plasmid p31RIT-12 (see Example 6b) is digested with SalI and XhoI. A 382 bp SalI~XhoI fragment is isolated from the gel by electroelution and ethanol precipitation. The fragment is further digested with BamHI and HgaI. A 591 bp BamHI-HgaI fragment, which contains the PH05 promoter region and the invertase signal sequence, is isolated.
Plazmid pJDB2O7/PHO5-TPA18 (glej evropsko patentno prijavo št. 143,O81) se prebavi z 3amHI in Xhol. 6.8 kb vektorski fragment izoliramo na preparativnem 0,6 % agaroznem gelu v Tris-acetatnem pufru, pH 8,2. DNA elektroeluiramo in precipitiramo z etanolom.Plasmid pJDB2O7/PHO5-TPA18 (see European Patent Application No. 143,081) was digested with 3amHI and XhoI. The 6.8 kb vector fragment was isolated on a preparative 0.6% agarose gel in Tris-acetate buffer, pH 8.2. The DNA was electroeluted and ethanol precipitated.
Vse DNA fragmente resuspendiramo v H^O pri koncentraciji 0,1 pmoi/^1. 0,2 pmola 591 bp BamHI-Hgal fragmenta, 0,2 pmola 312 bp Hgal-Pstl fragmenta, 0,2 pmola 1007 bp Pstl-Xhol fragmenta in 0,1 pmola 6,8 kb BamHI-XhoI vektorskega fragmenta vežemo 15 ur pri 15°C v 10 jil 50 mM Tris.HCl pH 7,5, 10 mM MgCl2, 5 mM DTT, 1 mM ATP in 400 enotah T4 DNA ligaze. En vesalne mešanice uporabljamo za transformiranjeAll DNA fragments were resuspended in H^O at a concentration of 0.1 pmoli/^1. 0.2 pmole of 591 bp BamHI-Hgal fragment, 0.2 pmole of 312 bp Hgal-Pstl fragment, 0.2 pmole of 1007 bp Pstl-Xhol fragment and 0.1 pmole of 6.8 kb BamHI-XhoI vector fragment were ligated for 15 hours at 15°C in 10 µl of 50 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 5 mM DTT, 1 mM ATP and 400 units of T4 DNA ligase. One part of the mixture was used for transformation.
ΌOh
E.ooli HB101 Ca ' celic. 12 amp‘ kolonij poberemo in gojimo v LB mediju, ki vsebuje 100 mg/1 ampicilina. DNA pripravimo po hitrem izolacijskem postopku /D.S. Holmes et al., Anal. Biochem. Ί14. 193 (19θ1)/. Na restrikcijskih prebavkih plazmida DNA. s Hinclll in EcoRI opazimo pričakovane restrikcijske fragmente. Plazmid DNA posameznega klona izberemo in ga omenjamo kot pJDB207/PH05-I-UPA.E.coli HB101 Ca ' cells. 12 amp‘ colonies are picked and grown in LB medium containing 100 mg/l ampicillin. DNA is prepared by a rapid isolation procedure /D.S. Holmes et al., Anal. Biochem. Ί14. 193 (19θ1)/. On restriction digestions of plasmid DNA. with Hinclll and EcoRI the expected restriction fragments are observed. Plasmid DNA of an individual clone is selected and referred to as pJDB207/PH05-I-UPA.
PRIMER 9: t-PA/u-PA hibridni plazminogeni aktivator s t-PA domenami A-verige in u-PA B-verige (primarna DNA konstrukcija)EXAMPLE 9: t-PA/u-PA hybrid plasminogen activator with t-PA A-chain and u-PA B-chain domains (primary DNA construct)
Drug pristop za konstrukcijo v okviru zlitja DNA zaporedij, ki kodirajo A-verigo t-PA in B-verigo u-PA v predhodno določeni legi, sestoji iz dveh stopenj: prvič, ustrezne restrikcijske fragmente s kodirnimi zaporedji povežemo. DNA pripravimo v E.coli in subkloniramo v M13, da dobimo enojnovlaknate šablone (template). V drugi stopnji prebitek nukleotidnih zaporedij odstranimo z in vitro mutagenezo. Natančna, uokvirjena zveza med t-PA A-verige in u-PA B-verige, je ob aktivacijskem mestu. Mutirano DNA subkloniramo v primernem ekspresijskem vektorju za kvas in celičnih linijah sesalcev.Another approach for the construction of a fusion of DNA sequences encoding the A-chain of t-PA and the B-chain of u-PA at a predetermined position consists of two steps: first, the appropriate restriction fragments are ligated to the coding sequences. The DNA is prepared in E. coli and subcloned into M13 to obtain single-stranded templates. In the second step, the excess nucleotide sequences are removed by in vitro mutagenesis. The precise, framed junction between the t-PA A-chain and the u-PA B-chain is located at the activation site. The mutated DNA is subcloned into a suitable expression vector for yeast and mammalian cell lines.
a) Izolacija DNA fragmenta, ki kodira t-PA A-verige:a) Isolation of the DNA fragment encoding the t-PA A-chain:
^tg plazmida pJDB207/PH05-I-TPA (glej primer 6) se prebavi z BamHI in PvuII. 1,7 kb BamHI-PvuII fragment ločimo na 0,8 % agaroznem gelu v Tris-borat-EDTA pufru, pH 8,3 DNA fragment vsebuje PHO5 promotor, invertazno signalno zaporedje in kodirno zaporedje zrelega t-PA do PvuII restrikcijskega mesta (primerjaj sl. 1; lege nukleotidov 1305-1310). DNA elektroeluiramo, precipitiramo z etanolom in resuspendiramo v H^O pri koncentraciji 0,1 pmolov/μΐ.^tg plasmid pJDB207/PH05-I-TPA (see Example 6) is digested with BamHI and PvuII. The 1.7 kb BamHI-PvuII fragment is separated on a 0.8% agarose gel in Tris-borate-EDTA buffer, pH 8.3. The DNA fragment contains the PHO5 promoter, the invertase signal sequence and the coding sequence of mature t-PA up to the PvuII restriction site (compare Fig. 1; nucleotide positions 1305-1310). The DNA is electroeluted, ethanol precipitated and resuspended in H^O at a concentration of 0.1 pmol/μΐ.
b) Izolacija DNA fragmenta, ki kodira u-PA B-verige:b) Isolation of the DNA fragment encoding u-PA B-chains:
Plazmid pCSl6/UPA (glej primer 7B) se prebavi z Bali (primerjaj sl. 3 in 4, lege nukleotidov 573-578) in Xhol. 868 bp Ball-Xhcl fragment izoliramo kot zgoraj in resuspendiramo v H^O pri koncentraciji 0,1 pmol/^il.Plasmid pCS16/UPA (see Example 7B) was digested with BalI (compare Figs. 3 and 4, nucleotide positions 573-578) and Xhol. The 868 bp Ball-Xhcl fragment was isolated as above and resuspended in H2O at a concentration of 0.1 pmol/µl.
c) Vezava fragmentov na vektorski fragment:c) Linking fragments to the vector fragment:
Plazmid pJDB207/PH05-TPAl8 (evropska patentna prijava št. 143,081) se prebavi z BamHI in Xhol. 6,7 kb vektorski fragment izoliramo na 0,8 % agaroznem gelu v Tris-acetatnem pufru, pH 8,2. DNA elektroeluiramo, precipitiramo z etanolom in resuspendiramo v H^O pri koncentraciji 0,1 pmol/^1.Plasmid pJDB207/PH05-TPAl8 (European Patent Application No. 143,081) was digested with BamHI and XhoI. The 6.7 kb vector fragment was isolated on a 0.8% agarose gel in Tris-acetate buffer, pH 8.2. The DNA was electroeluted, ethanol precipitated and resuspended in H^O at a concentration of 0.1 pmol/^l.
0,2 pmola 1,7 kb BamHI-PvuII fragmenta, 0,2 pmola 868 bp Ball-Xhol fragmenta in 0,1 pmol 6,7 kb BamHI-XhoI vektorskega fragmenta vežemo v 10 jil 60 mM Tris-HCl pH 7,5, 10 mM MgCl^, 5 mM DTT, 3,5 mM ATP in 400 enotah T^ DNA ligaze (Biolabs) pri 15°C 16 ur. Eno in 3 l.-ske alikvote ve žalne mešanice dodamo k 100 ^il Ca++ obdelanih E.coli HB101 celic. Transformiranje izpeljemo kot običajno.0.2 pmole of 1.7 kb BamHI-PvuII fragment, 0.2 pmole of 868 bp Ball-XhoI fragment and 0.1 pmole of 6.7 kb BamHI-XhoI vector fragment were ligated in 10 µl of 60 mM Tris-HCl pH 7.5, 10 mM MgCl^, 5 mM DTT, 3.5 mM ATP and 400 units of T^ DNA ligase (Biolabs) at 15°C for 16 hours. One and 3 µl aliquots of the ligation mixture were added to 100 µl of Ca++ treated E.coli HB101 cells. Transformation was carried out as usual.
Šest transformiranih, ampicilin odpornih kolonij raste v LB mediju, ki vsebuje 100 ^ig/1 ampicilina. Plazmid DNA pripravimo v smislu metode Holmsa in sodelavcev /Analyt. Biochem. 114, 193 (1981)/ in analiziramo po restrikcijskih prebavah z BamHI in Pstl. En klon s pričakovanimi restrikeijskimi fragmenti omenjamo kot pJDB207/PH05-I-TPA^UPA^.Six transformed, ampicillin-resistant colonies were grown in LB medium containing 100 µg/l ampicillin. Plasmid DNA was prepared according to the method of Holmes et al. (Analyt. Biochem. 114, 193 (1981)) and analyzed by restriction digestion with BamHI and PstI. One clone with the expected restriction fragments was designated pJDB207/PH05-I-TPA^UPA^.
PRIMER 10: u-PA/t-PA hibridni plazminogeni aktivator z domenami u-PA A-verige in t-PA B-verlge (primarna DNA konstrukcija)EXAMPLE 10: u-PA/t-PA hybrid plasminogen activator with u-PA A-chain and t-PA B-chain domains (primary DNA construct)
Primarna hibridna DNA konstrukcija obsega u-PA nukleotidna zaporedja od Smal mesta do EcoRl mesta (glej sl.4), vezana na t-PA nukleotidna zaporedja od Seal mesta (lege 940-945) do Xhol mesta, vpeljana ob legi 1800 preko Xhol povezovalca. Izhajajoče zaporedje hibridne DNA vsebuje prebitek nukleotidov, katere odstranimo z in vitro rautagenezo. Natančna, uokvirjena zveza u-PA A-verige in t-PA B-verige je ob aktivacijskem mestu.The primary hybrid DNA construct comprises the u-PA nucleotide sequences from the SmaI site to the EcoRI site (see Fig. 4), linked to the t-PA nucleotide sequences from the Seal site (positions 940-945) to the XhoI site, introduced at position 1800 via an XhoI linker. The resulting hybrid DNA sequence contains excess nucleotides, which are removed by in vitro rautagenesis. The precise, framed junction of the u-PA A-chain and the t-PA B-chain is at the activation site.
a) Izolacija DNA fragmenta, ki kodira u-PA A-verige:a) Isolation of the DNA fragment encoding the u-PA A-chain:
plazmida pCSl6/UPA se prebavi z EcoRI. Lepljive konce izhajajočih 3 fragmentov pretvorimo v tope konce z reakcijo vstavljanja s 7,5 enotami Klenowe DNA polimeraze (BRL) v prisotnosti 60 mM Tris.HCI pH 7,5, 10 mM MgCl?, 0,1mM dATP in 0,1 mM dTTP 30 min. pri 25°C. Reakcijo zaustavimo z dodatkom EDTA v končni koncentraciji 12,5 mM. DNA se nadalje prebavi s KpnI. Fragment 619 bp KpnI s topim /EcoRI/ koncem izoliramo na 1,5 % agaroznem gelu v Tris-borat-EDTA pufru, pH 8,3, elektroeluiramo in precipitiramo v etanolu.of plasmid pCS16/UPA is digested with EcoRI. The sticky ends of the resulting 3 fragments are converted to blunt ends by an insertion reaction with 7.5 units of Klenow DNA polymerase (BRL) in the presence of 60 mM Tris.HCl pH 7.5, 10 mM MgCl?, 0.1 mM dATP and 0.1 mM dTTP for 30 min. at 25°C. The reaction is stopped by the addition of EDTA to a final concentration of 12.5 mM. The DNA is further digested with KpnI. The 619 bp KpnI fragment with a blunt /EcoRI/ end is isolated on a 1.5% agarose gel in Tris-borate-EDTA buffer, pH 8.3, electroeluted and precipitated in ethanol.
b) Izolacija DNA fragmenta, ki kodira t-PA B-verige:b) Isolation of the DNA fragment encoding the t-PA B-chain:
^!g plazmida pJDB207/PH05-TPA18 se prebavi s Seal in Xhol. 860 bp fragment izoliramo na 1,2 % agaroznem gelu v Tris-borat-EDTA pufru, pH 8,3, elektroeluiramo in precipitiramo v etanolu.^!g of plasmid pJDB207/PH05-TPA18 is digested with ScaI and XhoI. The 860 bp fragment is isolated on a 1.2% agarose gel in Tris-borate-EDTA buffer, pH 8.3, electroeluted and precipitated in ethanol.
c) Vezava DNA fragmentov na pUCl8 izpeljan vektor:c) Ligation of DNA fragments to pUCl8 derived vector:
^g plazmida pCSl6/UPA (glej primer 7) se prebavi s KpnI in Xhol. Izhajajoči 2,7 kb fragment izoliramo na 0,8 % agaroznem gelu v Tris-borat-EDTA pufru, pH 8,3- DMA elektroeluiramo in precipitiramo v etanolu. Vse DNA fragmente resuspen diramo v H^O pri koncentraciji 0,1 pmola/^1.^g of plasmid pCSl6/UPA (see Example 7) is digested with KpnI and XhoI. The resulting 2.7 kb fragment is isolated on a 0.8% agarose gel in Tris-borate-EDTA buffer, pH 8.3. DMA is electroeluted and precipitated in ethanol. All DNA fragments are resuspended in H^O at a concentration of 0.1 pmole/^l.
0,2 pmola 619 bp Kpn-topi konec u-PA fragmenta, 0,2 pmola 860 bp Scal-Xhol t-PA fragmenta in 0,1 pmol 2,7 kb Kpnl-Xhol vektorskega framgenta vežemo, kot je opisano zgoraj (primer 9). Ca++ obdelane E.coli HB101 celice se transformirajo transformiranih, na ampicilin odpornih kolonij raste v LB mediju, ki je dopolnjen z ampicilinom (100 mg/1). DNA pripravimo v smislu Holmsa in sodelavcev (zgoraj) in analiziramo po restrikcijskih prebavah z EcoRI in Pstl. Posamezen klon s pričakovanimi restrikcijskimi fragmenti omenjamo kot pCSl6/UPAA/TPAB.0.2 pmoles of the 619 bp Kpn-blunt end u-PA fragment, 0.2 pmoles of the 860 bp Scal-XhoI t-PA fragment and 0.1 pmoles of the 2.7 kb KpnI-XhoI vector fragment were ligated as described above (Example 9). Ca++ treated E. coli HB101 cells were transformed and transformed, ampicillin resistant colonies were grown in LB medium supplemented with ampicillin (100 mg/l). DNA was prepared according to Holmes et al. (supra) and analyzed after restriction digestions with EcoRI and PstI. The individual clone with the expected restriction fragments was referred to as pCS16/UPAA /TPAB .
PRIMER 11: u-PA/t-PA hibridni plazminogeni aktivator z drugim kringle in katalitično B-verigo t-PA (primarna konstrukcija)EXAMPLE 11: u-PA/t-PA hybrid plasminogen activator with second kringle and catalytic B-chain of t-PA (primary construct)
Gen hibridnega plazminogenega aktivatorja, ki obsega DNA zaporedja rastnemu faktorju podobno (U)-domeno urokinaze, drugo kringle domeno (K2) t-PA in katalitično B-verigo t-PA, konstruiramo na sledeči način: Dva DNA restrikcijska fragmenta, ki kodirata u-PA domeno rastni faktor in t-PA K2 kringle oziroma B-verigo,vežemo in vstavimo v plazmid pCSl6. Izhajajoči klon imenujemo pCS16/UK2TPA' . Fragment, ki vsebuje zaporedja, ki kodirajo u-PA in t-PA. subkloniramo v M13. In vitro mutageneza poteka na eno-vlaknati DNA, da odstranimo prebitek DNA zaporedij pri zvezi med zaporedjema u-PA in t-PA.A hybrid plasminogen activator gene comprising the DNA sequences of the growth factor-like (U)-domain of urokinase, the second kringle domain (K2 ) of t-PA and the catalytic B-chain of t-PA is constructed as follows: Two DNA restriction fragments encoding the u-PA domain of the growth factor and the t-PA K2 kringle and B-chain, respectively, are ligated and inserted into plasmid pCS16. The resulting clone is designated pCS16/UK2 TPA' . The fragment containing the sequences encoding u-PA and t-PA is subcloned into M13. In vitro mutagenesis is performed on single-stranded DNA to remove excess DNA sequences at the junction between the u-PA and t-PA sequences.
^.g plazmida DCS16/UPA se prebavi z Ncol (lege nukleotidov 326-331, glej sl. 4). Lepljive konce restrikcijskih fragmentov zapolnimo v reakciji s 5 enotami Klenowe DNA polimeraze I (BRL) v prisotnosti 0,1 mM vsakega od dATP, dTTP, dCTP, dGTP, 60 mM Tris.HCl pH 7,5, 10 mM MgCl2 v 50 ^il 30 min. pri sobni temperaturi. Reakcijo zaustavimo z dodatkom EDTA v končni koncentraciji 12,5 mM. DNA precipitiramo v etanolu in se nadalje prebavi z Xhol. 3 kb fragment Xhol s topim koncem /NcoT/ izoliramo na 0,8 % agaroznem gelu v Tris-borat-EDTA, pH 8,3, elektroeluiramo in precipitiramo v etanolu. Ta fragment vsebuje pCSl6 vektor in kodirno zaporedje za u-PA rastni faktor domeno. 10 pg plazmida pJDB2O7/PHO5~TPA18 (evropska patentna prijava št. 143,081) se prebavi z BstXI /lege nukleotidov 577-588/. Linearni DNA fragment s 3’ preko visečimi konci inkubiramo z 10 enotami T^ DNA polimeraze (BRL) v 100 ^il 33 mM Tris-acetata pH 7,9, 66 mM kalijevega acetata, 10 mM magnezijevega acetata, 0,5 mM DTT in 0,1 mg/ml govejega serum albumina 2,5 min. pri 37°C. Inkubacijo nadaljujemo 35 min. pri 37°C v prisotnosti 0,1 mM vsakega od dATP, dCTP, dTTP, dGTP v celotnem volumnu 200 ^pl. DNA precipitiramo v etanolu in se nadalje prebavi z Xho'i. Fragment 1,2 kb s topim koncem /BstXI/-XhoI ločimo na 0,8 % agaroznem gelu, elektroeluiramo in precipitiramo v etanolu. Ta fragment vsebuje kodirno zaporedje za I<2 in B-verigo t-PA.^.g of plasmid DCS16/UPA is digested with NcoI (nucleotide positions 326-331, see Fig. 4). The sticky ends of the restriction fragments are filled in in a reaction with 5 units of Klenow DNA polymerase I (BRL) in the presence of 0.1 mM each of dATP, dTTP, dCTP, dGTP, 60 mM Tris.HCl pH 7.5, 10 mM MgCl2 in 50 ^il for 30 min. at room temperature. The reaction is stopped by the addition of EDTA to a final concentration of 12.5 mM. The DNA is precipitated in ethanol and further digested with XhoI. The 3 kb XhoI blunt-ended fragment /NcoT/ is isolated on a 0.8% agarose gel in Tris-borate-EDTA, pH 8.3, electroeluted and precipitated in ethanol. This fragment contains the pCSl6 vector and the coding sequence for the u-PA growth factor domain. 10 pg of plasmid pJDB2O7/PHO5~TPA18 (European Patent Application No. 143,081) was digested with BstXI (nucleotide positions 577-588). The linear DNA fragment with 3' overhanging ends was incubated with 10 units of T^ DNA polymerase (BRL) in 100 µl of 33 mM Tris-acetate pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DTT and 0.1 mg/ml bovine serum albumin for 2.5 min. at 37°C. Incubation was continued for 35 min. at 37°C in the presence of 0.1 mM each of dATP, dCTP, dTTP, dGTP in a total volume of 200 µl. The DNA was precipitated in ethanol and further digested with Xho'i. A 1.2 kb blunt-ended fragment (BstXI/-XhoI) was separated on a 0.8% agarose gel, electroeluted, and ethanol precipitated. This fragment contains the coding sequence for the I<2 and B-chain of t-PA.
0,2 pmola 1,2 kb t-PA fragmenta in 0,1 pmola 3 kb u-PA/vektorskega fragmenta (glej zgoraj) vežemo, kot je opisano. Alikvote vezaine mešanice uporabljamo za transformiranje primernih E.coli HB101 celic. Ampicilin-odporne kolonije izberemo na LB ploščah agarja, ki vsebujejo 100 mg/1 ampicilina. DNA pripravimo iz posameznih transformantov in analiziramo po Seal in Smal restrikcijskih prebavah. Klon, ki vsebuje 0,5 kb Seal in 1,55 kb Smal vezne fragmente, izberemo in ga omenjamo kot pCS15/UK2TPAB.0.2 pmoles of the 1.2 kb t-PA fragment and 0.1 pmoles of the 3 kb u-PA/vector fragment (see above) were ligated as described. Aliquots of the ligation mixture were used to transform suitable E. coli HB101 cells. Ampicillin-resistant colonies were selected on LB agar plates containing 100 mg/l ampicillin. DNA was prepared from individual transformants and analyzed by Smal and Smal restriction digestions. A clone containing the 0.5 kb Smal and 1.55 kb Smal ligation fragments was selected and referred to as pCS15/UK2 TPAB .
- 73 PRIMER 12: t-PA/u-PA hibridni plazminogeni aktivator z drugim kringle11 t-PA in katalitično B-verigo u-PA (primarna konstrukcija)- 73 EXAMPLE 12: t-PA/u-PA hybrid plasminogen activator with second kringle11 of t-PA and catalytic B-chain of u-PA (primary construct)
Gen hibridnega piazminogenega aktivatorja, ki obsega DNA zaporedja urokinazne rastnemu faktorju podobne (U) domene, drugi kringle (K2) t-PA in katalitično B-verigo u-PA, konstruiramo po metodi analogni tisti, ki je opisana v primeru 11.A hybrid plasminogen activator gene comprising the DNA sequences of the urokinase growth factor-like (U) domain, the second kringle (K2) of t-PA, and the catalytic B-chain of u-PA is constructed by a method analogous to that described in Example 11.
Konstrukcija plazmida pCS16/UKoUPA :Construction of plasmid pCS16/UKabout UPA:
'1 ........ .....11 li pg plazmida pCSl6/UPA se prebavi z Bglll in Ncol (lege nukleotidov 391-396 oziroma 326-331, glej sl. 4). Lepljive konce restrikcijskih fragmentov zapolnimo v reakciji s Klenowo DNA polimerazo I (BRL), kot je opisano zgoraj. 4,2 kb DNA fragment s topimi konci izoliramo na 0,8 % agaroznem gelu v Tris-acetatnem pufru, pH 8,2. DNA elektroeluiramo in precipitiramc v etanolu. Ta fragment vsebuje u-PA G--domeno in u-PA B-verigo, povezane na vektorsko molekulo.'1 ........ .....1 11 pg of plasmid pCS16/UPA is digested with BglII and NcoI (nucleotide positions 391-396 and 326-331, respectively, see Fig. 4). The sticky ends of the restriction fragments are filled in by a Klenow DNA polymerase I (BRL) reaction as described above. A 4.2 kb blunt-ended DNA fragment is isolated on a 0.8% agarose gel in Tris-acetate buffer, pH 8.2. The DNA is electroeluted and precipitated in ethanol. This fragment contains the u-PA G-domain and the u-PA B-chain linked to the vector molecule.
ug plazmida p31/PH05-TPA18 (evropska patentna prijava št. 143,081) se prebavi z Alul. 447 bp Alul fragment, ki vsebuje celotno K2 domeno t-PA, izoliramo na 1,5 % agaroznem gelu v Tris-borat-EDTA pufru, pH 8,3. DNA fragment elektroeluiramo in precipitiramo v etanolu.ug of plasmid p31/PH05-TPA18 (European Patent Application No. 143,081) is digested with Alul. A 447 bp Alul fragment containing the entire K2 domain of t-PA is isolated on a 1.5% agarose gel in Tris-borate-EDTA buffer, pH 8.3. The DNA fragment is electroeluted and ethanol precipitated.
0,2 pmolov 447 bp fragmenta in 0,1 praol 4,2 kb fragmenta vežemo. Alikvote vezalne mešanice uporabljamo za transformiranje primernih E.coli HB101 celic. Transformirane celice izberemo na LB agar ploščah s 100 mg/1 ampicilina. DNA pripravimo iz ampiciiin odpornih celic in analiziramo po EcoRI in Seal prebavah. Posamezen klon, ki kaže 551 bp EcoRI fragment in 403 bp Seal fragment, ima Alul fragment vstavljen g0.2 pmol of the 447 bp fragment and 0.1 pmol of the 4.2 kb fragment were ligated. Aliquots of the ligation mixture were used to transform suitable E. coli HB101 cells. Transformed cells were selected on LB agar plates with 100 mg/l ampicillin. DNA was prepared from ampicillin-resistant cells and analyzed by EcoRI and Seal digestions. A single clone showing a 551 bp EcoRI fragment and a 403 bp Seal fragment had an Alul fragment inserted into it.
v pravilni orientaciji. Ta klon omenjamo kot pCSl6/UK2UPA .in the correct orientation. We refer to this clone as pCSl6/UK2 UPA .
PRIMER 13: Kloniranje primarnih hibridnih DNA konstrukcij v Ml3mp18EXAMPLE 13: Cloning of primary hybrid DNA constructs into Ml3mp18
A) Kloniranje p,JDB207/PHO5-I-TPAAUPAB BamHI fragmenta vA) Cloning of the p,JDB207/PHO5-I-TPAA UPAB BamHI fragment into
M13mpl8M13mpl8
1,5 ^ig pJDB207/PH05-I-TPAAUPAB (primerjaj primer 9), dobljenega iz hitre DNA priprave, se prebavi z 9 U BamHI (Boehringer) v 20 jil 10 mM Tris.HCl pH 7,5 6 mM MgCl2,1.5 µg of pJDB207/PH05-I-TPAA UPAB (compare Example 9), obtained from the rapid DNA preparation, is digested with 9 U BamHI (Boehringer) in 20 µl of 10 mM Tris.HCl pH 7.5 6 mM MgCl2 ,
100 mM NaCl, 6 mM merkaptoetanola pri 37°C eno uro. Po dodatku 1 ^1 RNaze (Serva, 1 mg/ml), inkubiranju 15 min. pri 37°C in fenolizaciji, izoliramo 2,5 kb vkljueek na 0,8 % pneparativnem agaroznem gelu. DNA ekstrahiramo z elektroeluiranjem in precipitiramo.100 mM NaCl, 6 mM mercaptoethanol at 37°C for one hour. After addition of 1 µl RNase (Serva, 1 mg/ml), incubation for 15 min. at 37°C and phenolization, a 2.5 kb insert was isolated on a 0.8% preparative agarose gel. The DNA was extracted by electroelution and precipitated.
^tg M13mpl8 (P.F) režemo z BamHI, obdelamo s telečjo črevesno alkalno fosfatazo in izoliramo 7,3 kb vektorski fragment na 0,7 % pneparativnem agaroznem gelu. DMA elektroeluiramo in precipitiramo.^tg M13mpl8 (P.F) was cut with BamHI, treated with calf intestinal alkaline phosphatase, and the 7.3 kb vector fragment was isolated on a 0.7% preparative agarose gel. The DMA was electroeluted and precipitated.
100 pmolov H13mpl8 BamHI rezanega vektorja in 200 pmolov BamHI TPAAUPAB vključka vežemo v 10 jil 50 mM Tris.HCl pH' 7,5, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, 0,5 ^ig želatine s 400 U T^ DNA ligaze 7 ur pri 15°C. Po inkubaciji pri 65°C 10 min.100 pmol of H13mpl8 BamHI cut vector and 200 pmol of BamHI TPAA UPAB insert were ligated in 10 µl of 50 mM Tris.HCl pH' 7.5, 10 mM MgCl2 , 10 mM DTT, 2 mM ATP, 0.5 µg gelatin with 400 UT^ DNA ligase for 7 hours at 15°C. After incubation at 65°C for 10 min.
uporabljamo 5 ^il te vezalne mešanice za transformacijo E.coli JM101 primernih celic v smislu s priročnikatfM13 Cloning and Sequencing Handbook”, ki ga je izdal Amersham. 36 brezbarvnih plakov poberemo in pripravimo eno-vlaknato in replikativno obliko (RF) DNA. Pri analizi RF-DNA kažejo vsi kloni vključke pravilne velikosti po prebavi z BamHI. Fragmenti pravilne velikosti po prebavi z EcoRI in Pstl kažejo, da so DNA vključki v vseh klonih v napačni orientaciji (eno-vlaknata šablona DNA je ne-kodirajoče vlakno). Enega od teh klonov omenjamo kot Δ R mpl8/BaraHI/TPA /UPA in ga uporabljamo za delecijsko mutagenezo.5 µl of this ligation mixture is used to transform E.coli JM101 suitable cells according to the“ M13 Cloning and Sequencing Handbook” published by Amersham. 36 colorless plaques are picked and single-stranded and replicative form (RF) DNA is prepared. In RF-DNA analysis, all clones show inserts of the correct size after digestion with BamHI. Fragments of the correct size after digestion with EcoRI and PstI indicate that the DNA inserts in all clones are in the wrong orientation (the single-stranded DNA template is the non-coding strand). One of these clones is referred to as Δ R mpl8/BaraHI/TPA /UPA and is used for deletion mutagenesis.
B. Kloniranje pCS16/UPAATPAB KpnI-Hindlll fragmenta v M13mpl8B. Cloning of the pCS16/UPAA TPAB KpnI-HindIII fragment into M13mpl8
1,5 yg pCS16/UPAATPAB (primerjaj primer 10), dobljen iz hitre DNA priprave, se prebavi z 12 U KpnI v 20 ^il 10 raM Tris.HCl pH 7,5, 6 mM MgCl^, 6 mM merkaptoetanola pri 37°C eno uro. Po dodatku 1 yil 1 M NaCl, se DNA prebavi z 12 U Hindlll pri 37°C v eni uri. 1,5 kb fragment izoliramo na 0,8 % preparativnem agaroznem gelu. DNA ekstrahiramo z elektroeluiranjem in precipitiramo.1.5 μg of pCS16/UPAA TPAB (compare Example 10), obtained from a rapid DNA preparation, is digested with 12 U KpnI in 20 μl of 10 mM Tris.HCl pH 7.5, 6 mM MgCl^, 6 mM mercaptoethanol at 37°C for one hour. After addition of 1 μl of 1 M NaCl, the DNA is digested with 12 U HindIII at 37°C for one hour. The 1.5 kb fragment is isolated on a 0.8% preparative agarose gel. The DNA is extracted by electroelution and precipitated.
0,5 M13mp 18 (RF) se prebavi s KpnI in Hindlll. Izoliramo 7,3 kb vektorski fragment na 0,7 % preparativnem agaroznem gelu. DMA elektroeluiramo in precipitiramo.0.5 M13mp 18 (RF) is digested with KpnI and HindIII. The 7.3 kb vector fragment is isolated on a 0.7% preparative agarose gel. The DMA is electroeluted and precipitated.
100 fmolov M13mpl8 KpnI-Hindlll rezanega vektorja in 200 fmolov KpnI-Hindlll vključka vežemo v 10 ^j1 50 mM Tris.HCl pH 7,5, 10 mM MgCl^, 10 mM DTT, 2 mM ATP, 0,5 ^ig želatine s 400 U Tjj DNA ligaze 7 ur pri 15°C. Reakcijo zaustavimo z inkubacijo pri 65°C 10 min. 5 ^1 te vezalne mešanice uporabljamo za transformiranje E.coli JM101 primernih celic. Deset brezbarvnih plakov poberemo in pripravimo eno-vlaknato in replikativno obliko (RF) DNA. Pri analizi RF-DNA kažejo vsi kloni pravilno velikost vključkov in pravilno velikost fragmentov. Enega od teh klonov omenjamo kot mp18/KpnI-HindIII/UPAA/TPA® in ga uporabljamo za delecijsko mutagenezo.100 fmoles of M13mpl8 KpnI-HindIII cut vector and 200 fmoles of KpnI-HindIII insert were ligated in 10 µl of 50 mM Tris.HCl pH 7.5, 10 mM MgCl^, 10 mM DTT, 2 mM ATP, 0.5 µg gelatin with 400 U Tjj DNA ligase for 7 hours at 15°C. The reaction was stopped by incubation at 65°C for 10 min. 5 µl of this ligation mixture was used to transform E.coli JM101 competent cells. Ten colorless plaques were picked and single-stranded and replicative form (RF) DNA was prepared. In RF-DNA analysis, all clones showed the correct size of the inserts and the correct size of the fragments. One of these clones was designated mp18/KpnI-HindIII/UPAA /TPA® and was used for deletion mutagenesis.
7b7b
C. Kloniranje pCS16/UK2TPAB KpnI-Hindlll fragmenta v M13mpl3C. Cloning of the pCS16/UK2 TPAB KpnI-HindIII fragment into M13mpl3
1,5 ^ig pCS1 o/UK^TPA® (primerjaj primer 11) dobljen iz hitre DNA priprave, se prebavi z 12 U KpnI (Boehringer) v 20 ^1 10 mM Tris-HCl pH 7,5, 6 mM MgCl2, 6 mM merkaptoetanola pri 37°C eno uro. Po dodatku 1 jal 1 M NaCl, se DNA prebavi z 12 U Hindlll pri 37°C eno uro. Izoliramo 1,5 kb fragment na 0,8 % preparativnem agaroznem gelu. DNA ekstrahiramo z elektro eluiranjem in precipitiramo.1.5 µg of pCS1 o/UK^TPA® (compare Example 11) obtained from the rapid DNA preparation is digested with 12 U KpnI (Boehringer) in 20 µl of 10 mM Tris-HCl pH 7.5, 6 mM MgCl2 , 6 mM mercaptoethanol at 37°C for one hour. After addition of 1 µl of 1 M NaCl, the DNA is digested with 12 U HindIII at 37°C for one hour. A 1.5 kb fragment is isolated on a 0.8% preparative agarose gel. The DNA is extracted by electroelution and precipitated.
0,5 M13mpl8 (RF) se prebavi s KpnI in Hindlll. Izoliramo 7,3 Mb vektorski fragment na 0,7 % preparativnem agaroznem gelu. DNA elektroeluiramo in precipitiramo.0.5 M13mpl8 (RF) is digested with KpnI and HindIII. A 7.3 Mb vector fragment is isolated on a 0.7% preparative agarose gel. The DNA is electroeluted and precipitated.
100 fmolov M13mpl8 KpnI-Hindlll reza vektorja in 200 fmolov KpnI-Hindlll vključka vežemo v 10 50 mM Tris.HCl pH 7,5, 10 mM MgCl2, 10 mti DTT, 2 mM A1P, 0,5 pg želatine s 400 U DNA ligaze 7 ur pri 15°C. Reakcijo zaustavimo z inkubacijo pri 65°C 10 min. 5 yil te vezalne mešanice uporabljamo za transformiranje E.coli JM101 primernih celic. Sedem brezbarvnih plakov poberemo in pripravimo eno-vlaknato in replikativno obliko (RF) DNA. Pri analizi RF-DNA kažejo vsi kloni pravilno velikost vključkov in pravilno velikost fragmentov. Enega teh kionov omenjamo kot mpl8/KpnI-HindlII/UK2TPAB in ga uporabljamo za delečijsko mutagenezo.100 fmol of M13mpl8 KpnI-HindIII cut vector and 200 fmol of KpnI-HindIII insert were ligated in 10 50 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 10 mM DTT, 2 mM A1P, 0.5 μg gelatin with 400 U DNA ligase for 7 h at 15°C. The reaction was stopped by incubation at 65°C for 10 min. 5 μl of this ligation mixture was used to transform E. coli JM101 competent cells. Seven colorless plaques were picked and single-stranded and replicative form (RF) DNA was prepared. In RF-DNA analysis, all clones showed the correct size of the inserts and the correct size of the fragments. One of these clones was designated mpl8/KpnI-HindIII/UK2 TPAB and was used for deletion mutagenesis.
D. Kloniranje pCS16/UK?UPAB KpnI-Hindlll fragmenta v M13mpl8D. Cloning of the pCS16/UK? UPAB KpnI-HindIII fragment into M13mpl8
1,5 PCS16/UK2UPAB (primerjaj primer 12), dobljen iz hitre DNA priprave, se prebavi z 12 U KpnI v 20 ^il 10 m.M Tris.HCl pH 7,5, 6 mM MgCl2> 6 mM merkaptoetanola pri 37°C eno uro. Po dodatku 1 ^il 1 M NaCl se DNA prebavi z 12 U Hindlll pri 37°C v eni uri. izoliramo 1,7 kb fragment na 0,8 % preparativnem agaroznem gelu. DMA ekstrahiramo s elektroeluira njem in precipitiramo.1.5 PCS16/UK2 UPAB (compare Example 12), obtained from a rapid DNA preparation, is digested with 12 U KpnI in 20 µl of 10 mM Tris.HCl pH 7.5, 6 mM MgCl2 > 6 mM mercaptoethanol at 37°C for one hour. After addition of 1 µl of 1 M NaCl, the DNA is digested with 12 U HindIII at 37°C for one hour. A 1.7 kb fragment is isolated on a 0.8% preparative agarose gel. DMA is extracted by electroelution and precipitated.
0,5 pg i-i13mpl8 (RF) se prebavi s KpnI in Hindlll. Izoliramo 7,3 kb vektorski fragment na 0,7 % preparativnem agaroznem gelu. DNA elektroeluiramo in precipitiramo.0.5 pg of i-i13mpl8 (RF) was digested with KpnI and HindIII. The 7.3 kb vector fragment was isolated on a 0.7% preparative agarose gel. The DNA was electroeluted and precipitated.
100 fmolov M13mpl8 KpnI-Hindlll reza vektorja in 200 fmolov KpnI-Hindlll vključka vežemo v 10 ^il 50 mM Tris.HCl pH 7,5, 10 mM MgCl2, 10 mil DTT, 2 mM ATP, 0,5 ^ig želatine s 400 U Tjj DMA ligaze 7 ur pri 15°C. Reakcijo zaustavimo z inkubacijo pri 65°C 10 min. 5 ^il te vezalne mešanice uporabimo za transformiranje E.coli JM101 primernih celic. Deset brezbarvnih plakov poberemo in pripravimo eno-vlaknato in replikativno obliko (RF) DNA. Pri analizi RF-DNA kažejo vsi kloni pravilno velikost vkljuekov in pravilno velikost fragmentov.100 fmol of M13mpl8 KpnI-HindIII cut vector and 200 fmol of KpnI-HindIII insert were ligated in 10 µl of 50 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 10 µl DTT, 2 mM ATP, 0.5 µg gelatin with 400 U Tjj DMA ligase for 7 hours at 15°C. The reaction was stopped by incubation at 65°C for 10 min. 5 µl of this ligation mixture was used to transform E.coli JM101 competent cells. Ten colorless plaques were picked and single-stranded and replicative form (RF) DNA was prepared. In RF-DNA analysis, all clones showed the correct size of the inserts and the correct size of the fragments.
ββ
Enega teh klonov omenjamo kot mpl8/KpnI-HindIII/UI<2UPA· in ga uporabljamo za delecijsko mutagenezo.One of these clones is referred to as mpl8/KpnI-HindIII/UI<2 UPA· and is used for deletion mutagenesis.
PRIMER 14: Deiecijska mutageneza primarnih hibridnih DNA konstrukcijEXAMPLE 14: Deletion mutagenesis of primary hybrid DNA constructs
A) Splošni protokol delecijske mutagenezeA) General deletion mutagenesis protocol
a) Fosforilacija mutagenega primerja:a) Phosphorylation of the mutagenic primer:
Za mutagenezo fosforiliramo 200 pmolov mutagenega primerja v 20 pl 50 mM Tris-HCl pH 7,5, 10 mM MgCl2, 5 mM DTT, 0,1 mM spermidina, 0,1 mM EDTA, ki vsebuje 1 1 10 mM ATP in uporabi 8 U T^ polinukleotid kinaze (Boehringer, 8 U /p 1ri Q ' 'J segreva£For mutagenesis, 200 pmol of mutagenic primer was phosphorylated in 20 µl of 50 mM Tris-HCl pH 7.5, 10 mM MgCl2 , 5 mM DTT, 0.1 mM spermidine, 0.1 mM EDTA containing 1 µl of 10 mM ATP and 8 µL of polynucleotide kinase (Boehringer, 8 U/µl , heated to 100 µL).
Po inkubaciji pri 37 C eno uro. reakcijo zaustavimo njem na 65°C 10 min.After incubation at 37°C for one hour, the reaction was stopped at 65°C for 10 min.
Za hibridizacijski skrining, 20 pmolov mutagenega •.32 primerja fosforiliramo kot zgoraj z uporabo 30 /iCi ‘S -P-ATP (3000 Ci/mmol: Amersham International; kot edini vir ATP). Primer razredčimo s 3,5 ml 6 x SSC in uporabljamo direktno kot vzorec.For hybridization screening, 20 pmol of mutagenic •.32 primer was phosphorylated as above using 30 µCi of ‘S-P-ATP (3000 Ci/mmol: Amersham International; as sole source of ATP). The primer was diluted with 3.5 ml of 6 x SSC and used directly as a template.
b) Renaturiranje mutagenega primerja in splošnega sekvenčnega primerja z eno-vlaknato šablonob) Renaturation of the mutagenic primer and the general sequencing primer with a single-stranded template
0,2 pmola eno-vlaknate šablone inkubiramo z 20 pmoli fosforiliranega mutagenega oligodeoksiribonukleotidnega primerja (10 pmolov//il) in 10 pmoli splošnega M13 sekvenčnega primerja v 10 pl 20 mM Tris.HCI pK 7,5, 10 mM MgCl2, 50 mM0.2 pmoles of single-stranded template were incubated with 20 pmoles of phosphorylated mutagenic oligodeoxyribonucleotide primer (10 pmoles/µl) and 10 pmoles of general M13 sequence primer in 10 µl of 20 mM Tris.HCl pK 7.5, 10 mM MgCl2 , 50 mM
NaCl, 1 mM DTT pri 95°C 5 min. Raztopino pustimo počasi ohlajati na sobno temperaturo v obdobju 30 min.NaCl, 1 mM DTT at 95°C for 5 min. The solution was allowed to cool slowly to room temperature over a period of 30 min.
c) Reakcija podaljšanja-vezanjac) Extension-coupling reaction
H gornji nenaturacijski mešanici dodamo 10 /il encimdNTP (dATP, dGTP, dTTP, dCTP) raztopine, ki vsebuje 1 /il pufra /0,2 M Tris.HCI pH 7,5, 0,1 MgCl2, 0,1 M DTT/, 4 /ji 2,5 mM dNTP mešanice, 1 /il 10 mM ATP, 0,5 /il T^ DNA ligaze (Biolabs. 400 U//ii), 0,67 /il Klenove DNA polimeraze (BRL, 2,99 U//il). Mešanico inkubiramo pri 15°C eno uro in nato inkubiramo pri 8° - 9°C 16 ur. Reakcijo zaustavimo z inkubiranjem pri 65°C 10 min.To the above denaturation mixture, add 10 µl of dNTP enzyme (dATP, dGTP, dTTP, dCTP) solution containing 1 µl of buffer (0.2 M Tris.HCl pH 7.5, 0.1 MgCl2 , 0.1 M DTT), 4 µl of 2.5 mM dNTP mixture, 1 µl of 10 mM ATP, 0.5 µl of T^ DNA ligase (Biolabs. 400 U//ii), 0.67 µl of Klenow DNA polymerase (BRL, 2.99 U//il). The mixture is incubated at 15°C for one hour and then incubated at 8° - 9°C for 16 hours. The reaction is stopped by incubation at 65°C for 10 min.
d) Transformacija vezalne mešaniced) Transformation of the binding mixture
Vezalno mešanico razredčimo na 1:20 in 1:200 s TE: po 1 ^il in 5 ^1 vsake od teh razredčenih raztopin uporabljamo za transformiranje 0,2 ml ne-reparacijskega soja. E.coli BMH 71~l8mutS /BMH71-l8(£Ylac-proAB), thi, supE, £’laciq, Z«TlT 15, proA+B+/ primernih celic. Konstrukcijo E.coli BMH71-l8mutS (BMH71-18, mut S215::Tnio) je opisal Kramer in sodel. /Celi 38, 879-887 (1984)/. Po transfekciji so celice trate oskublje ne z reparacijskim sojem E.coli JM101 zato, da se do skrajnosti zmanjša izpostavljanje faga mutatorskemu fenotipu nereparacijskih sojev /P. Carter, H. Bedouelle and G. Winter, Nucl. Acids Res. J_3> 4431-4443 ( 1 985)/.The ligation mixture was diluted 1:20 and 1:200 with TE: 1 µl and 5 µl of each of these dilutions were used to transform 0.2 ml of the non-repairing strain. E. coli BMH 71-18mutS /BMH71-18(£Ylac-proAB), thi, supE, £'laciq , Z«TlT 15, proA+ B+ / suitable cells. The construction of E. coli BMH71-18mutS (BMH71-18, mut S215::Tnio) was described by Kramer et al. /Cells 38, 879-887 (1984)/. After transfection, the cells were overexpressed with the repairing strain E. coli JM101 in order to minimize exposure of the phage to the mutator phenotype of the non-repairing strains /P. Carter, H. Bedouelle and G. Winter, Nucl. Acids Res. J_3> 4431-4443 ( 1 985)/.
e) Skrinlng fagove) Phage screening
100 plakov, ki izhajajo iz transficirane DNA, prenesemo z zobotrebcem v YT plošče in zrastejo kot kolonije inficiranih bakterij v 15 - 18 urah. Popivnanje kolonij je prilagojeno po Grunstein in Hogness /Proč. Mati. Acad. Sci. USA 72, 39613965 (1985)/. Nitrocelulozni filter (Millipore S.A., Cat. No. HAWP 090, velikost por 0,45 pm) namestimo na ploščo kolonije za 10 min. pri sobni temperaturi. Filtre denaturiramo z 0,5 K NaOH, nevtraliziramo z 1 M Tris.HCl pH 7,5 in nato obdelamo z močno slano raztopino (0,5 M Tris.HCl pH 7,5 + 1,5 M NaCl). Filtre pečemo v vakuumu 30 min. pri 80°C, predhibridiziramo v 100 ml 10 x Denhardtove raztopine (D.T. Denhardt, Biochem. Biophvs. Res. Commun. 23, 641-646), 6 χ SSC in 0,2 % SDS v zaprti plastični vreči 15 min.100 plaques derived from transfected DNA are transferred with a toothpick to YT plates and grow as colonies of infected bacteria within 15 - 18 hours. Colony pick-up is adapted from Grunstein and Hogness /Proc. Mati. Acad. Sci. USA 72, 39613965 (1985)/. A nitrocellulose filter (Millipore S.A., Cat. No. HAWP 090, pore size 0.45 pm) is placed on the colony plate for 10 min. at room temperature. The filters are denatured with 0.5 K NaOH, neutralized with 1 M Tris.HCl pH 7.5 and then treated with a strong salt solution (0.5 M Tris.HCl pH 7.5 + 1.5 M NaCl). The filters are baked in a vacuum for 30 min. at 80°C, prehybridized in 100 ml of 10 x Denhardt's solution (D.T. Denhardt, Biochem. Biophvs. Res. Commun. 23, 641-646), 6 χ SSC and 0.2% SDS in a sealed plastic bag for 15 min.
n „ Ltci.n „ The pilots.
izpiramo v ml vzorca, nibridizacijski skrining predhibridizirane ml 6 x SSC 1 minuto in nato hibridiziramo ki vsebuje P-označen mutageni primer, 30 izperemo minuti in tipom inwashed in ml of sample, hybridization screening prehybridized ml 6 x SSC containing P-labeled mutagenic primer, washed for 30 minutes and type and
Hibridizirane filtre trikrat sobni temperaturi, v celoti 2 Dobro razlikovanje med divjim po kratkem izpiranju (5 min.) 0,1 % SDS.Hybridized filters three times at room temperature, in total 2 Good discrimination between wild type after short wash (5 min.) 0.1% SDS.
filtre v 3,5 minut.filters in 3.5 minutes.
v 100 ml 6 x SSC pri nato avtoradiografiramo mutiranimi fagi dobimo pri 60°C v 100 ml 0,1 x SSC +in 100 ml 6 x SSC at 60°C then autoradiographed the mutated phages in 100 ml 0.1 x SSC +
f) Potrdilo delecijske mutacije v pozitivnih klonih, dobljenih iz hibridizacijef) Confirmation of deletion mutation in positive clones obtained from hybridization
Fage iz pozitivnih klonov prenesemo z zobotrebcem v 1 ml 2 χ YT, segrevamo pri 70°C 20 min., da ubijemo bakterije in nato 100 ^il te suspenzije fagov inokuliramo v 1,5 ml sveže rastoče E.coli JM101 kulture 0,45). Kulture močno stresamo (300 obr./min.) pri 37°C 4 ure. Fage na zalogi in replikativnc obliko DNA pripravimo iz pozitivnih klonov /J. Messing, Methods in Enzymology, 101 , 21—7δ ( 1983)/. DNA iz mutant (po delecijski mutagenezi) analiziramo s primernimi restrikcijskimi encimi in jih primerjamo z restrikcijskimi fragmenti divjega tipa (pred delecijsko mutagenezo) DNA. Po potrdilu z restrikcijsko analizo, DNA iz ene pravilne mutante piakovno čistimo. Mutacije nadalje potrdimo po restrikcijski analizi in sekvenciranjem z uporabo metode verižnega zaključevalca /F. Sanger, S. Niclen and. A.H. Coulson, Proč. Natl. Acad. Sci. USA 74, 5463-5467 (1977)/.Phages from positive clones are transferred with a toothpick into 1 ml of 2 x YT, heated at 70°C for 20 min. to kill the bacteria and then 100 µl of this phage suspension is inoculated into 1.5 ml of freshly grown E. coli JM101 culture 0.45). The cultures are shaken vigorously (300 rpm) at 37°C for 4 hours. Phage stocks and replicative DNA are prepared from positive clones /J. Messing, Methods in Enzymology, 101 , 21—7δ ( 1983)/. DNA from mutants (after deletion mutagenesis) is analyzed with appropriate restriction enzymes and compared with restriction fragments of wild-type (before deletion mutagenesis) DNA. After confirmation by restriction analysis, DNA from one correct mutant is purified by plate purification. Mutations are further confirmed by restriction analysis and sequencing using the chain terminator method /F. Sanger, S. Niclen and. A.H. Coulson, Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)/.
η μη μ
Β) Delecijska mutageneza na mp18/BamHI/TPA^UPA1 (glej sl. 23)Β) Deletion mutagenesis on mp18/BamHI/TPA^UPA1 (see Fig. 23)
Delecijsko mutagenezo izpeljemo, kot je opisano v splošnem protokolu. Pozitivne klone, ki jih dobimo iz hibridizacije, potrdimo pri restrikeijski analizi. 333 bp odstranimo pri delecijski mutagenezi iz BamHI fragmenta. Restrikeijska analiza z BamHI potrdi 2150 bp fragment. Nadaljna restrikcijska analiza z EcoRI da 660, 416, 287, 230 bp fragmente na mutantah namesto 660, 472, 416 in 287 fragmentov, vidnih na divjem tipu. Analiza s Pstl kaže dva fragmenta, v velikosti 611 in 414 bp za mutante. Divji tip DNA kaže tri fragmente 622, 611 in 414 bps. En mutiran klon, ki ima pravilno strukturo, omenjamo kot mp18/BamHI/M0TPA^UPA®.Deletion mutagenesis is performed as described in the general protocol. Positive clones obtained from hybridization are confirmed by restriction analysis. 333 bp are removed by deletion mutagenesis from the BamHI fragment. Restriction analysis with BamHI confirms a 2150 bp fragment. Further restriction analysis with EcoRI gives 660, 416, 287, 230 bp fragments in the mutants instead of the 660, 472, 416 and 287 fragments seen in the wild type. Analysis with Pstl shows two fragments, 611 and 414 bp in size for the mutants. The wild type DNA shows three fragments of 622, 611 and 414 bps. One mutant clone that has the correct structure is referred to as mp18/BamHI/MOTPA^UPA®.
DNA zaporedje pri zvezi med t-PA A-verige in u-PA B-verige potrdimo s sekvenčno metodo verižnega zaključevalca, ki ima sekvenčni primer zaporedja 5 ’ CAGAGCCCCCCCGGTGC 3’.The DNA sequence at the junction between t-PA A-chain and u-PA B-chain is confirmed by the chain terminator sequencing method, which has a sequence primer of the sequence 5' CAGAGCCCCCCCCGGTGC 3'.
Ta primer je komplementaren kodirnemu vlaknu u-PA (682-666).This primer is complementary to the u-PA coding strand (682-666).
C) Delecijska mutageneza na mpl8/KpnI-HindIII/UPA^TPA® n ' .I-I. I — . I II 11—, m. ι I..N·.· I MIH» I I —I I —.........C) Deletion mutagenesis on mpl8/KpnI-HindIII/UPA^TPA® n ' .I-I. I — . I II 11—, m. ι I..N·.· I MIH» I I —I I —.........
(glej sl. 24)(see Fig. 24)
Delecijsko mutagenezo izpeljemo, kot je opisano v splošnem protokolu. Pozitivne klone, ki jih dobimo iz hibridizacije, potrdimo pri restrikeijski analizi s Pstl. V mutantah opazimo 467 bp pas v primerjavi z divjim tipom, kateri da 544 bp fragment. En mutiran klon, ki ima pravilno strukturo, omenjamo kot rap18/KpnI-HindIIi/M0UPAATPA^. Delecijo potrdimo s sekvenčno metodo verižnega zaključevalca z uporabo sekvenčnega primerja zaporedja 5’CAAAGATGGCAOCCTGC 3’.Deletion mutagenesis was performed as described in the general protocol. Positive clones obtained from hybridization were confirmed by restriction analysis with PstI. A 467 bp band was observed in the mutants compared to the wild type, which yielded a 544 bp fragment. One mutant clone with the correct structure was designated rap18/KpnI-HindIIi/M0UPAA TPA^. The deletion was confirmed by chain terminator sequencing using the sequence primer 5'CAAAGATGGCAOCCTGC 3'.
Ta primer je komplementaren kodirnemu vlaknu t-PA (1062-1046).This primer is complementary to the t-PA coding strand (1062-1046).
D. Delecijska mutageneza na mpl8/i^nI-HindIII/UKnTPA®D. Deletion mutagenesis on mpl8/i^nI-HindIII/UKn TPA®
........... ............. . .................. .Ul I|£ (glej sl. 25)........... ............. . .................. .Ul I|£ (see Fig. 25)
Delečijsko mutagenezo izpeljemo, kot je opisano v splošnem protokolu. Pozitivne klone, ki jih dobimo iz hibridizacije, potrdimo pri restrikcijski analizi s KpnI-Hindlll,Deletion mutagenesis was performed as described in the general protocol. Positive clones obtained from hybridization were confirmed by restriction analysis with KpnI-HindIII,
EcoRI in Pstl. Fragmente, ki jih dobimo, so · .....ηρ»- --------------------- ----------------------------' ’ ΊEcoRI and Pstl. The fragments obtained are · .....ηρ»- -------------------- ----------------------------' ’ Ί
KpnI-Hindlll EcoRI Pstl divji tip mutanta divji tip mutanta divji tip mutantaKpnI-HindIII EcoRI Pstl wild type mutant wild type mutant wild type mutant
1475 bp 1284 , bp 542 bp 351 bp 622 bp brez 622 bp traku1475 bp 1284 , bp 542 bp 351 bp 622 bp no 622 bp band
Za mutante ugotovimo pravilno velikost inserta in fragmentov. Enega izmed mutantnih klonov s pravilno strukturo naB vajamo kot mpl8/KpnI-HindIII/MOUK2TPA . Izpustitev verificiramo z metodo verižno-terminatorskega sekvenciranja ob uporabi sekvencirnega primerja s sekvenco (zaporedjem).The correct insert and fragment sizes are determined for the mutants. One of the mutant clones with the correct naB structure is designated as mpl8/KpnI-HindIII/MOUK2 TPA . The deletion is verified by chain-terminator sequencing using a sequencing primer with the sequence (sequence).
5’ CCCAGTGCCTGGGCACTGGGGTTCTGTGCTGTG 3’·5' CCCAGTGCCTGGGCACTGGGGTTCTGTGCTGTG 3'·
Ta primer je komplementaren h kodirni verigi t-PA (853821).This primer is complementary to the coding strand of t-PA (853821).
- 83 E) peleciJskg , . mutageneza na mpl8/KpnI-HindIII/UKgUPAB (glej sl. 26) n - - V konstrukciji UKgUPA sodelujeta dve ločeni delecijski mutaciji:- 83 E) peleciJskg , . mutagenesis at mpl8/KpnI-HindIII/UKg UPAB (see Fig. 26) n - - Two separate deletion mutations are involved in the construction of UKgUPA:
Prvo deleči jskomutagenezo izvedemo tako, kot je opisano v splošnem protokolu. Pozitivni kloni, dobljeni pri hibridizaciji, so potrjeni z restrikcijsko analizo z EcoRl. V mutantih opazimo trakove 549, 416, 351 bp, v primeri z divjim tipom, ki daje fragmente 549, 452 in 416 bp. En mutanten klon gThe first divisive mutagenesis was performed as described in the general protocol. Positive clones obtained from hybridization were confirmed by restriction analysis with EcoRI. In the mutants, bands of 549, 416, 351 bp were observed, in the case of the wild type, which gave fragments of 549, 452 and 416 bp. One mutant clone g
s pravilno strukturo navajamo kot mpl8/KpnI-HindIII/M0UK2UPA 1. Delecijo verificiramo z metodo verižno-terminatorskega sekvenciranja ob uporabi sekvencirnega primerja s sekvenco5’ CCCAGTGCCTGGGCACTGGGGTTCTGTGCTGTG 3’.with the correct structure is given as mpl8/KpnI-HindIII/M0UK2 UPA 1. The deletion is verified by the chain-terminator sequencing method using a sequencing primer with the sequence5 ' CCCAGTGCCTGGGCACTGGGGTTCTGTGCTGTG 3'.
Primer je komplementaren h kodirni verigi t-PA, (853821).The primer is complementary to the coding strand of t-PA, (853821).
V drugi stopnji delecijske mutageneze izvedemo delecijo istočasno z uvedbo točkovne mutacije. Delecijsko mutagenezo izvedemo tako, kot je opisano v splošnem protokolu. Pozitivni kloni, dobljeni pri hibridizaciji, so potrjeni z restrikcijsko analizo z EcoRl. V mutantih opazimo trakove 416, 351, 259 bp, v primeri z divjim tipom, ki daje fragmente 549, 416 in 351 bp. En mutanten klon s pravilno strukturo navajamo kot mpl8/KpnI-HindIII/M0UK2UPA®. Delecijo verificiramo z metodo verižno-terminatorskega sekvenciranja ob uporabi sekvencirnega primerja s sekvencoIn the second stage of deletion mutagenesis, the deletion is performed simultaneously with the introduction of a point mutation. Deletion mutagenesis is performed as described in the general protocol. Positive clones obtained from hybridization are confirmed by restriction analysis with EcoRI. In the mutants, bands of 416, 351, 259 bp are observed, in the case of the wild type, which gives fragments of 549, 416 and 351 bp. One mutant clone with the correct structure is designated as mpl8/KpnI-HindIII/M0UK2 UPA®. The deletion is verified by the chain-terminator sequencing method using a sequencing primer with the sequence
5’ CAGAGCCCCCCCGGTGC 3’.5’ CAGAGCCCCCCCGGTGC 3’.
Primer je komplementaren h kodirni verigi u-PA (682-666)The primer is complementary to the coding strand of u-PA (682-666)
PRIMER 15;:· Kloniranje hibridnih t-PA/u-PA cDNA konstruktov v kvasni ekspresijski vektor pJDB207EXAMPLE 15; Cloning of hybrid t-PA/u-PA cDNA constructs into yeast expression vector pJDB207
A) Kloniranje TPA^UPA^ hibridnega gena v pJDB207A) Cloning of the TPA^UPA^ hybrid gene into pJDB207
RF-DNA pripravimo za mpl8/BamHI/M0TPA^UPA® s hitrim postopkom izolacije DNA /D.S. Holmes in M. Quingley, Anal. Biochem. 114, 192-197 (1981)/.RF-DNA was prepared for mpl8/BamHI/MOTPA^UPA® by the rapid DNA isolation procedure (D.S. Holmes and M. Quingley, Anal. Biochem. 114, 192-197 (1981)).
RF-DNA ( 1,5 pg) prebavimo z 9 U BamHI v 20 pl 10 mM Tris.HCl pH 7,5, 6 mM MgCl2, 100 mM NaCl, 6 mM merkaptoetanola eno uro pri 37°C. Po dodatku 1 pl RNaze (1 mg/ml) in 10 minutnem inkubiranju pri 37°C izoliramo 2.1 kb insert na 0,7% preparativnega agaroznega gela. DNA insert ekstrahiramo z elektroelucijo in oborimo v etanolu.RF-DNA (1.5 pg) was digested with 9 U BamHI in 20 µl of 10 mM Tris.HCl pH 7.5, 6 mM MgCl2 , 100 mM NaCl, 6 mM mercaptoethanol for one hour at 37°C. After addition of 1 µl of RNase (1 mg/ml) and incubation for 10 minutes at 37°C, the 2.1 kb insert was isolated on a 0.7% preparative agarose gel. The DNA insert was extracted by electroelution and precipitated in ethanol.
,5 pg p.PDB207/PH05-I-TPA^UPA? razrežemo z BamHI, obdelamo z alkalno fosfatazo telečjega črevesa ter izoliramo 6.7 kb vektor. Po elektroeluciji oborimo vektor DNA..5 pg of p.PDB207/PH05-I-TPA^UPA? was digested with BamHI, treated with calf intestinal alkaline phosphatase, and the 6.7 kb vector was isolated. After electroelution, the vector DNA was precipitated.
100 fmolov pJDB207/PH05-I-TPAAUPAB BamHI razrezanega vektorja, 200 fmolov TPA UPA Inserta povežemo v 10 ^il 50 mM100 fmoles of pJDB207/PH05-I-TPAA UPAB BamHI digested vector, 200 fmoles of TPA UPA Insert were ligated into 10 µl of 50 mM
Tris.HCI pH 7,5, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, 0,5 yig' želatine s 400 U T^ DNA ligaze v teku 8 ur pri 15°C. Reakcijo ustavimo z inkubiranjem pri 65°C v teku 10 minut. 5 yil te vezivne zmesi uporabimo za transformacijo E.coli HB101 Ca celic /M. Dagert in S.D. Ehrlich, Gene 6, 23-28 ( 1979)/· 12 ampRkolonij poberemo in pripravimo DNA s hitrim izolacijskim postopkom. Pri analizi DNA 5 klonov pokaže tako pravilno velikost insertov kot tudi pravilno orientacijo. En klon gojimo na 100 ml LB medija, ki vsebuje 100 mg/ml ampicilina. Izoliramo plazmid DNA in ga označimo kot pJDB2O7/PHO5-I-MOTPAAUPAB.Tris.HCl pH 7.5, 10 mM MgCl2 , 10 mM DTT, 2 mM ATP, 0.5 μg gelatin with 400 UT^ DNA ligase for 8 hours at 15°C. The reaction is stopped by incubation at 65°C for 10 minutes. 5 μl of this ligation mixture is used to transform E.coli HB101 Ca cells /M. Dagert and SD Ehrlich, Gene 6, 23-28 (1979)/· 12 ampR colonies are picked and DNA is prepared by a rapid isolation procedure. On DNA analysis, 5 clones show both the correct insert size and the correct orientation. One clone is grown in 100 ml of LB medium containing 100 mg/ml ampicillin. Plasmid DNA is isolated and designated pJDB2O7/PHO5-I-MOTPAA UPAB .
B) Kloniranje M0UPAATPAB, MOUKgTPA6 IN MOUKgUPA6 genskih insertov v plazmid pCSl6B) Cloning of the M0UPAA TPAB , MOUKgTPA6 AND MOUKgUPA6 gene inserts into the plasmid pCSl6
RF-DNA pripravimo za mpl8/KpnI-HindIII/M0UPAATPAB, mpl8/KpnI-HindIII/M0UK2TPAB, mpl8/KpnI-HindIII/MOUK2UPABs hitrim postopkom izolacije DNA.RF-DNA was prepared for mpl8/KpnI-HindIII/M0UPAA TPAB , mpl8/KpnI-HindIII/M0UK2TPAB , mpl8/KpnI-HindIII/MOUK2UPAB by a rapid DNA isolation procedure.
Tri RF-DNA (~1.5 ^ug) vsakokrat prebavimo z 12 U KpnI in 12 U Hindlll v 20 ^1 10 mM Tris.HCI pH 7,5, 6 mM MgCl2, 6mM merkaptoetanola v teku 1 ure pri 37°C. Dodamo 1 jal 1 M NaCl ter vse DNA nadalje prebavimo z 12 U Hindlll. Po dodatku 1 ml RNaze (1 mg/ml) in inkubiranju v teku 10 minut pri 37°C, izoliramo vsakokrat 1.4 kb inserte na 0,8% preparativnega agaroznega gela. DNA inserte ekstrahiramo z elektroelucijo in oborimo v etanolu.Three RF-DNAs (~1.5 µg) were each digested with 12 U KpnI and 12 U HindIII in 20 µl 10 mM Tris.HCl pH 7.5, 6 mM MgCl2 , 6 mM mercaptoethanol for 1 hour at 37°C. 1 µl 1 M NaCl was added and all DNA was further digested with 12 U HindIII. After adding 1 ml RNase (1 mg/ml) and incubating for 10 minutes at 37°C, 1.4 kb inserts were each isolated on a 0.8% preparative agarose gel. The DNA inserts were extracted by electroelution and precipitated in ethanol.
Tri ^ug pCS16/UPA prebavimo s KpnI in Hind III in izoliramo 2.7 vektorski fragment. Po elektroeluciji oborimo vektorThree µg of pCS16/UPA are digested with KpnI and Hind III and the 2.7 vector fragment is isolated. After electroelution, the vector is precipitated
DNA v etanolu.DNA in ethanol.
- 86 100 fmolov pCSl6 KpnI-Hindlll razrezanega vektorja,- 86 100 fmoles of pCS16 KpnI-HindIII digested vector,
200 fmolov KpnI-Hindlll razrezanih insertnih fragmentov povežemo v 10 fl 50 mM Tris.HCl pH 7.5, 10 mM MgCl2, 10 mM DTT,200 fmol of KpnI-HindIII digested insert fragments were ligated into 10 µl of 50 mM Tris.HCl pH 7.5, 10 mM MgCl2 , 10 mM DTT,
2mM ATP, 0.5 fg želatine s 400 U DNA ligaze v teku 8 ur pri2mM ATP, 0.5 fg gelatin with 400 U DNA ligase for 8 hours at
15°C. Reakcijo prekinemo z inkubacijo v teku 10 minut pri 65°C 5 jil te vezivne zmesi uporabimo za transformacijo celic E.coli15°C. The reaction was terminated by incubation for 10 minutes at 65°C. 5 µl of this binding mixture was used to transform E.coli cells.
HB101 Ca2+.HB101 Ca2+ .
x Rx R
Sest amp kolonij poberemo od vsakih izmed treh vezav.Six amp colonies are picked from each of the three bindings.
DNA pripravimo s hitrim izolacijskim postopkom. Pri analizi DNA s KpnI-Hindlll opazimo insertne trakove s pravilno velikostjo. En klon izmed vsakega od treh vezav gojimo v 100 ml LB medija, ki vsebuje 100 mg/ml ampicilina. Plazmidne DNA, izvedene iz ιηρΙδ/ΙφηΙ-ΗίηάΙΙΙ/ΜΟυΡΑ^ΡΑ®, mpl8/KpnI-HindIII/MOUK2TPAB in. mpl8/KpnI-HindIII/MOUK2UPAB, -izoliramo in označimo kot ρΟείδ/ΜΟΟΡΑ^ΡΑ®, PCS16/M0UK2TPAB in pCS16/MOUK2UPAB.DNA was prepared by a rapid isolation procedure. Insertion bands of the correct size were observed when DNA was analyzed with KpnI-HindIII. One clone from each of the three ligations was grown in 100 ml of LB medium containing 100 mg/ml ampicillin. Plasmid DNAs derived from πηρΙδ/ΙφηΙ-ΗίηάΙΙΙ/ΜΟυΡΑ^ΡΑ®, mpl8/KpnI-HindIII/MOUK2TPAB and mpl8/KpnI-HindIII/MOUK2UPAB were isolated and designated as ρΟείδ/ΜΟΟΟΡΑ^ΡΑ®, PCS16/M0UK2 TPAB and pCS16/MOUK2 UPAB .
C)) Kloniranje MOUPA^PA2, MOUK2TPAB in MOUKzUPA2 genskih insertov ' '—————————- —---, - .........C)) Cloning of MOUPA^PA2 , MOUK2 TPAB and MOUKzUPA2 gene inserts ''—————————- —---, - .........
v pJDB2O7 pg pJDB207/PH05-I-UPA prebavimo s 15 U Seal in 15 Uin pJDB2O7 pg pJDB207/PH05-I-UPA is digested with 15 U Seal and 15 U
Xhol (Boehringer v 50 pl 10 mM Tris.HCl pH 7,5, 6 mM MgClg,Xhol (Boehringer in 50 µl 10 mM Tris.HCl pH 7.5, 6 mM MgClg,
150 mM NaCl, 6 mM merkaptoetanola v teku 1 ure pri 37°C. Po dodatku 1 ^1 RNaze (1 mg/ml) izoliramo 6.7 kb vektorski fragment. Po elektroeluciji oborimo vektor DNA.150 mM NaCl, 6 mM mercaptoethanol for 1 hour at 37°C. After addition of 1 µl RNase (1 mg/ml), a 6.7 kb vector fragment is isolated. After electroelution, the vector DNA is precipitated.
Po 15 fig PCS16/MOUPAATPAB, pCSl6/MOUK2TPAB, pCSl6/15 figs each PCS16/MOUPAA TPAB , pCSl6/MOUK2 TPAB , pCSl6/
M0UK2UPAB inkubiramo pri 37°C za 1 uro s 30 U Xhol v 200 pl 10 mM Tris.HCl pH 8, 6 mM MgCl2, 150 mM NaCl, 6 mM merkaptoetanola, ekstrahiramo z enakim volumnom fenola-kloroforma ter oborimo v etanolu. Oborjene, z Xhol razrezane, pCS16/M0UPAATPAB, pCS16/MOUK2TEAB in pCS16/MOUK2UPAB DNA, vsakokrat resuspendiramo v 150 jil 10 mM Tris.HCI pH 7.5, 6 mM MgCl^, 150 mM NaCl, 6 mM merkaptoetanola, 1.5 jig homidijevega (etidijevega) bromida, inkubiramo pri 37°C za 40 minut z 12 U Seal (delna prebava) ter ekstrahiramo z enakim volumnom fenola, zatem pa še z enakim volumnom kloroforma-izoamilalkohola (50:1). 1.2 kb fragmente vsakokrat izoliramo na 1% preparativnega agaroznega gela. DNA ekstrahiramo z elektroelucijo in oborimo.M0UK2 UPAB was incubated at 37°C for 1 hour with 30 U Xhol in 200 µl of 10 mM Tris.HCl pH 8, 6 mM MgCl2 , 150 mM NaCl, 6 mM mercaptoethanol, extracted with an equal volume of phenol-chloroform and precipitated in ethanol. The precipitated, XhoI-cut, pCS16/MOUPAA TPAB , pCS16/MOUK2TEAB and pCS16/MOUK2UPAB DNAs were each resuspended in 150 µl of 10 mM Tris.HCl pH 7.5, 6 mM MgCl^, 150 mM NaCl, 6 mM mercaptoethanol, 1.5 µg ethidium bromide, incubated at 37°C for 40 minutes with 12 U Seal (partial digestion) and extracted with an equal volume of phenol, then with an equal volume of chloroform-isoamyl alcohol (50:1). The 1.2 kb fragments were each isolated on a 1% preparative agarose gel. The DNA was extracted by electroelution and precipitated.
100 fmolov pJDB207/PH05-I-UPA Scal-Xhol razrezanega100 fmol pJDB207/PH05-I-UPA Scal-XhoI cut
A B vektorja in 200 fmolov Xho-Scal razrezanega pCSl6/MOUPA TPA , pCS16/MOUK2TPAB oz‘. PCS16/MOUK2UPAB 1.2 kb insertov, povežemo v 10 2*1 50 mM Tris. HCI pH 7,5, 10 mM MgCl2, 10 mM DTT, 2 mM AT.P, 0,5 ji g želatine s 400 U T^ DNA ligaze v teku 16 ur pri 15°C. Reakcijo ustavimo z inkubacijo pri 65°C v teku 10 minut. 5 jjI te vezivne zmesi uporabimo za transformacijo celic E.coliAB vector and 200 fmol of Xho-Scal digested pCS16/MOUPA TPA , pCS16/MOUK2 TPAB or PCS16/MOUK2UPAB 1.2 kb inserts, were ligated in 10 2*1 50 mM Tris. HCl pH 7.5, 10 mM MgCl2, 10 mM DTT, 2 mM ATP, 0.5 µg gelatin with 400 UT^ DNA ligase for 16 hours at 15°C. The reaction was stopped by incubation at 65°C for 10 minutes. 5 µl of this ligation mixture was used to transform E. coli cells
HB101 Ca2+.HB101 Ca2+ .
Λ RΛ R
Sest amp kolonij poberemo od vsake izmed teh treh vezi. DNA pripravimo s hitrim izolacijskim postopkom. Restrikcijska liza DNA kaže pravilno velikost insertnih trakov. En klon od vsake izmed treh povezav gojimo v 100 ml LB medija, ki vsebujeSix amp colonies are picked from each of these three linkages. DNA is prepared by a rapid isolation procedure. Restriction lysis of the DNA shows the correct size of the insert bands. One clone from each of the three linkages is grown in 100 ml of LB medium containing
AA
100 ^ig/ml ampicilina. Plazmidne DNA, izvedene iz pSCl6/MOUPA TPAB, pCS16/M0UK2TPAB, pCS16/M0UK2UPAB označujemo kot pJDB207/ PH05-I-MOUPAATPAB, pJDB207/PH05-I-M0UK2TPAB oz.pJDB207/ΡΗΟ5-Ιmouk2upab.100 ^ig/ml ampicillin. Plasmid DNAs derived from pSCl6/MOUPA TPAB , pCS16/M0UK2TPAB, pCS16/M0UK2UPAB are designated as pJDB207/ PH05-I-MOUPAA TPAB , pJDB207/PH05-I-M0UK2TPAB or pJDB207/ΡΗΟ5-Ιmouk2 upab .
PRIMER 16: Transformacija Saccharomyces cerevisiae GRF18 in priprava ekstraktov celic kvasovkEXAMPLE 16: Transformation of Saccharomyces cerevisiae GRF18 and preparation of yeast cell extracts
Plazmide pJDB207/PHO5-I-MOTPAAUPAB, pJDB207/PH05-IMOUPAATPAB, pJDB207/PHO5-I-MOUK2TEAB in pJDB207/PHO5-I-MOUK2βPlasmids pJDB207/PHO5-I-MOTPAA UPAB , pJDB207/PH05-IMOUPAA TPAB , pJDB207/PHO5-I-MOUK2TEAB and pJDB207/PHO5-I-MOUK2β
UPA vsakokrat uvedemo v Saccharomyces cerevisiae soj GRF18?ob uporabi transformacijskega protokola,ki so opisali Hinnen et al. /P>roc. N,ati. Acad. Sci. USA 75, 1929 ( 1978)/. Po 5 jjg vsake od plazmidnih DNA dodamo k 100 jal sferoplastne suspenzije ter zmes obdelamo s polietilenglikolom. Sferoplaste pomešamo z 10 ml regeneracijskega agarja in'jih sadimo v ’ ploščo z minimalI nim medijem kvasa brez levcina. Po 3-dnevni inkubaciji pri 30°C dobimo okoli 200 transformiranih celic.UPA is introduced into Saccharomyces cerevisiae strainGRF18 using the transformation protocol described by Hinnen et al. /Proc. Nat. Acad. Sci. USA 75, 1929 (1978)/. 5 µg of each plasmid DNA is added to 100 µl of spheroplast suspension and the mixture is treated with polyethylene glycol. The spheroplasts are mixed with 10 ml of regeneration agar and plated on a plate with minimal yeast medium without leucine. After 3 days of incubation at 30°C, approximately 200 transformed cells are obtained.
Poberemo po eno kolonijo od vsake izmed kvasnih transformacij .Pick one colony from each of the yeast transformations.
Različne kolonije označujemo kot:We refer to different colonies as:
Saccharomyces cerevisiae GRF18/pJDB207/PHO5-I-M0TPAAUPA®Saccharomyces cerevisiae GRF18/pJDB207/PHO5-I-M0TPAA UPA®
Saccharomyces cerevisiae GRFl8/pJDB2O7/PHO5-I-MOUPAATPA^Saccharomyces cerevisiae GRFl8/pJDB2O7/PHO5-I-MOUPAA TPA^
Saccharomyces cerevisiae GRFl8/pJDB2O7/PHO5-I-MQUK2TPA^Saccharomyces cerevisiae GRFl8/pJDB2O7/PHO5-I-MQUK2TPA^
Saccharomyces cerevisiae GRF18/pJDB207/PH05-I-MOUK2UPA®Saccharomyces cerevisiae GRF18/pJDB207/PH05-I-MOUK2UPA®
Kvasne celice gojimo pri 30°C v 20 ml HE.17 medija (8,4 g Yeast Nitrogen Base (Difco), 10 g L-asparagina (Sigma), 1 g L-histidina (Sigma), 40 ml 50% glukoze na 1 1 raztopine), v lYeast cells were grown at 30°C in 20 ml HE.17 medium (8.4 g Yeast Nitrogen Base (Difco), 10 g L-asparagine (Sigma), 1 g L-histidine (Sigma), 40 ml 50% glucose per 1 l of solution), in l
ml-ski erlenmajerici ob stresanju 24 ur, dokler ne dosežemo J gostote 8-10x10 celic/ml. Celice centrifugiramo in ponovno suspendiramo v 10 ml 0,9 % NaCl. 2 ml ponovno suspendiranih celic uporabimo za inokulacijo 50 ml šibko P^ minimalnega medija (kot je opisano v evropski patentni prijavi št. 143081),ml Erlenmeyer flasks with shaking for 24 hours until a density of 8-10x10 cells/ml is reached. The cells are centrifuged and resuspended in 10 ml 0.9% NaCl. 2 ml of the resuspended cells are used to inoculate 50 ml of weak P^ minimal medium (as described in European Patent Application No. 143081),
- 8.9 k čemur dodamo 10 g/1 L-asparagina (Sigma), , in 10 g/1 L-histidina (Sigma), v 250 ml-skih erlenmajericah. Inkubacija je pri 30°C in 250 obr/min.- 8.9 to which 10 g/l L-asparagine (Sigma), and 10 g/l L-histidine (Sigma), are added, in 250 ml Erlenmeyer flasks. Incubation is at 30°C and 250 rpm.
Celice iz 10 ml šibko P^ minimalnega medija zberemo po 48 urah s centrifugiranjem pri 3000 obr/min. v teku 10 minut v Falcon 2070 epruvetah. Celice enkrat speremo z 10 ml šibko P^ medija in centrifugiramo. Celično peletko suspendiramo v liznem pufru / 66 mM kalijevega fosfata s pH 7,4, 4mM Zwittergent (Calbiochem.)/. K celični suspenziji dodamo 8 g steklenih kroglic (s premerom 0,5-0,75 mm) in stekleno paličico, nakar stresamo suspenzijo v Vortex mešalniku (Scientific Instruments Inc. USA) s polno hitrostjo 4x2 minuti z intervali po 2 minuti na ledu. Več kot 90% celic se razpoči pri tem postopku. Drobce celic in steklene kroglice sedimentiramo s centrifugiranjem v teku 5 minut pri 3000 obr/min. pri 4°C.Vrhnji sloj (supernatant) uporabimo za določitev PA aktivnosti in za očiščenje in izolacijo PA.Cells from 10 ml of weak P^ minimal medium were collected after 48 hours by centrifugation at 3000 rpm for 10 minutes in Falcon 2070 tubes. The cells were washed once with 10 ml of weak P^ medium and centrifuged. The cell pellet was suspended in lysis buffer /66 mM potassium phosphate pH 7.4, 4 mM Zwittergent (Calbiochem.)/. 8 g of glass beads (0.5-0.75 mm diameter) and a glass rod were added to the cell suspension, and the suspension was vortexed (Scientific Instruments Inc. USA) at full speed for 4x2 minutes with 2-minute intervals on ice. More than 90% of the cells were disrupted during this procedure. Cell debris and glass beads were sedimented by centrifugation for 5 minutes at 3000 rpm. at 4°C. The upper layer (supernatant) is used for the determination of PA activity and for the purification and isolation of PA.
PRIMER 17: Uvedba hibridnih PA kodirnih sekvenc v celično ekspresijski vektor sesalcevEXAMPLE 17: Introduction of hybrid PA coding sequences into a mammalian cell expression vector
A BA B
A) Uvedba (insert) UPA TPA , popolne, hibridne kodirne sekvenceA) Introduction (insert) of UPA TPA, complete, hybrid coding sequence
RF DNA mpl8/KpnI-HindIII/M0UPA^TPA^ narežemo na Smal mestu,ki se nahaja ravno nad pričetkom kodirne sekvence in vežemo na SacI linker (CGAGCTCG). Zatem plazmid razrežemo s SacI, ki zareže v položaju vezanih linkerjev in na naravnem Seal mestu v t-PA-izvedenem deležu sekvence, ki kodira za hibrid PA. Manjšega izmed obeh nastalih fragmentov očistimo preko agaroz- 9C nega gela in ga vežemo na Sacl-razrezan pCGA44 (glej primer 4), transfor miran v E.coli HB101,in DNA iz kandidatnih klonov testiramo z EcoRI. Klon s pričakovanim restrikcijskim vzorcem navajamo kot pCGCl/UPAATPAB.The RF DNA mpl8/KpnI-HindIII/M0UPA^TPA^ is cut at the SmaI site just upstream of the coding sequence and ligated to the SacI linker (CGAGCTCG). The plasmid is then cut with SacI, which cuts at the position of the ligated linkers and at the natural ScaI site in the t-PA-derived portion of the sequence coding for the PA hybrid. The smaller of the two resulting fragments is purified on an agarose-9C gel and ligated to SacI-cut pCGA44 (see Example 4), transformed into E. coli HB101, and DNA from candidate clones is probed with EcoRI. The clone with the expected restriction pattern is designated pCGCI/UPAA TPAB .
gMr.
B) Uvedba UK?TPA hibridne kodirne sekvenceB) Introduction of UK? TPA hybrid coding sequence
RF DNA mp18/KpnI-HindIII/MOUK2T'PAB narežemo na Smal mestu, ki se nahaja ravno nad pričetkom kodirne sekvence in vežemo na SacI kot zgoraj. Po razrezanju s SacI kloniramo na stali majhni fragment v Sacl-razrezan pCGA44, kot je opisano zgoraj, ter klon s pričakovanim restrikcijskim vzorcem navajamo kot pCGC2/UK2TPAB .The RF DNA mp18/KpnI-HindIII/MOUK2 T'PAB was cut at the SmaI site just upstream of the coding sequence and ligated with SacI as above. After cutting with SacI, the resulting small fragment was cloned into SacI-cut pCGA44 as described above, and the clone with the expected restriction pattern was designated pCGC2/UK2 TPAB .
gMr.
C) Uvedba UK2UPA hibridne kodirne sekvenceC) Introduction of UK2 UPA hybrid coding sequence
RF DNA mpl8/KpnI-HindIII/M0UK2UPAB narežemo na Smal mestu nad u-PA kodirno sekvenco ter pri Xhol mestu pod kodirno sekvenco (v vektor DNA). Lepljivi konec DNA fragmenta zapolnimo z uporabo E.coli DNA polimeraze I (glej primer 5D) fRF DNA mpl8/KpnI-HindIII/M0UK2 UPAB is cut at the SmaI site above the u-PA coding sequence and at the XhoI site below the coding sequence (into the DNA vector). The sticky end of the DNA fragment is filled in using E.coli DNA polymerase I (see Example 5D) f
SacI linkerje povežem» na topih koncih, DNA razrežemo s SacI, manjšega izmed obeh nastalih fragmentov pa očistimo preko agaroznega gela in kloniramo v Sacl-razrezan pCGA44. Klon s pričakovanim EcoRI restrikcijskim vzorcem navajamo kot pCGC3/UK2UPAB.SacI linkers are ligated at the blunt ends, the DNA is digested with SacI, the smaller of the two resulting fragments is purified through an agarose gel and cloned into SacI-digested pCGA44. The clone with the expected EcoRI restriction pattern is designated as pCGC3/UK2 UPAB .
A BA B
D Uvedba TPA UPA , popolne, kodirne sekvenceD Introduction of TPA UPA, complete, coding sequences
Stopnja 1: RF DNA mpl8/BamHI/M0TPAAUPAB razrežemo z 'BamHI in manjšega (približno 2.1 kb) izmed fragmentov kloniI ramo v BamHI narezan pJDB207/PH05-I-TPAAUPAB(glej primer 9) vektor. Pravilno orientacijo izberemc),s prebavi janjem s Hind-ž aStep 1: The RF DNA mpl8/BamHI/MOTPAA UPAB is cut with BamHI and the smaller (approximately 2.1 kb) fragment is cloned into the BamHI cut pJDB207/PH05-I-TPAA UPAB (see Example 9) vector. The correct orientation is selected by digestion with Hind-
III in en pravilen plazmid imenujemo pJDB207/PHO5-I-MOTPA UPAB.III and one correct plasmid is called pJDB207/PHO5-I-MOTPA UPAB .
STOPNJA 2: 600 bp Sacl-Narl fragment iz ptNC.UC (glej primer 3) in ^>1350 bp Narl-Xhol fragment iz pJDB207/PH05-IA BSTEP 2: 600 bp SacI-Narl fragment from ptNC.UC (see example 3) and ^>1350 bp Narl-Xhol fragment from pJDB207/PH05-IA B
TPA UPA izoliramo in kloniramo v Sacl-Xhol razrezan pCSl6 (glej primer 7) vektor.1.9 kb insert potrdimo s prebavljanjem s Sacl-Xhol in EcoRI. En pravilen plazmid imenujemo PCS16/M0TPAAUPAB.TPA UPA was isolated and cloned into the SacI-XhoI cut pCS16 (see Example 7) vector. The 1.9 kb insert was confirmed by digestion with SacI-XhoI and EcoRI. One correct plasmid was designated PCS16/M0TPAA UPAB .
Stopnja 3: Plazmid pCS16/MOTPAAUPAB razrežemo na Xhol mestu, i ' lociranem pod u-PA kodirno sekvenco,ter lepljive konce zapolnimo z uporabo E.coli DNA polimeraze I. SacI linkerje vežemo na topih koncih in DNA razrežemo s SacI. Manjšega izmed obeh fragmentov očistimo preko agaroznega gela in kloniramo v Sacl-razrezan pBR4a (glej primer 5) vektorski fragment. Pravilna orientacija in pravilna velikost insertov so potrjeni s prebavljanjem z BamHI oz.SacI. En pravilen plazmid označimo kot pCGC4a/TPAAUPAB.Step 3: Plasmid pCS16/MOTPAA UPAB is cut at the XhoI site located below the u-PA coding sequence, and the sticky ends are filled in using E. coli DNA polymerase I. SacI linkers are ligated at the blunt ends and the DNA is cut with SacI. The smaller of the two fragments is purified on an agarose gel and cloned into the SacI-cut pBR4a (see Example 5) vector fragment. The correct orientation and size of the inserts are confirmed by digestion with BamHI or SacI. One correct plasmid is designated pCGC4a/TPAA UPAB .
PRIMER 18: Konstrukcija nadaljnjih hibridnih PA kodirnih sekvenc in njihova uvedba v ekspresijski vektor celice sesalcevEXAMPLE 18: Construction of further hybrid PA coding sequences and their introduction into a mammalian cell expression vector
A) Kloniranje pCGC4a/TPAAUPAB fragmenta v M13mpl8 yug pCGC4a/TPAAUPAB (glej primer 17) prebavimo zA) Cloning of the pCGC4a/TPAA UPAB fragment into M13mpl8 yug pCGC4a/TPAA UPAB (see example 17) is digested with
U SacI (Boehringer) v 20 yd 10 mM Tris-HCl pH 7,5, 6 mM MgCl2, 6 mM merkaptoetanola pri 37°C v 1 uri. ^1.9 kb fragment izoliramo na 0,7% preparativnega agaroznega gela.U SacI (Boehringer) in 20 μl of 10 mM Tris-HCl pH 7.5, 6 mM MgCl2 , 6 mM mercaptoethanol at 37°C for 1 hour. The ~1.9 kb fragment was isolated on a 0.7% preparative agarose gel.
9.29.2
DNA ekstrahiramo z elektroelucijo in oborimo.DNA is extracted by electroelution and precipitated.
0,5 pg M13mpl8 (RF) prebavimo s SacI. 7-3 kb vektorski fragment izoliramo na 0,7% preparativnega agaroznega gela. DNA elektroeluiramo in oborimo.0.5 pg of M13mpl8 (RF) was digested with SacI. The 7-3 kb vector fragment was isolated on a 0.7% preparative agarose gel. The DNA was electroeluted and precipitated.
100 fmolov M13mpl8 SacI razrezanega vektorja in 200 fmolov SacI inserta vežemo v 10 pl 50 mM Tris-HCl pH 7,5, mM MgCl2, 10 mM DTT, 2 mM ATP, 0,5 jig želatine s 400 U DNA ligaze v teku 7 ur pri 15°C. Reakcijo ustavimo z inkubiranjem pri 65°C 10 minut. 5 pl te vezivne zmesi uporabimo za transformacijo E.coli JM101 kompetentnih celic. Poberemo šest brezbarvnih plakov (ploščic) in pripravimo enovlaknato in replikativno obliko (RF) DNA. Pri analizi R-DNA kažejo štirje kloni pravilno velikost insertov in pravilno orientacijo. Enega izmed teh klonov navajamo kot mpl8/SacI/TPA UPA (BC).100 fmoles of M13mpl8 SacI digested vector and 200 fmoles of SacI insert were ligated in 10 µl of 50 mM Tris-HCl pH 7.5, mM MgCl2 , 10 mM DTT, 2 mM ATP, 0.5 µg gelatin with 400 U DNA ligase for 7 hours at 15°C. The reaction was stopped by incubation at 65°C for 10 minutes. 5 µl of this ligation mixture was used to transform E. coli JM101 competent cells. Six colorless plaques were picked and single-stranded and replicative form (RF) DNA was prepared. Four clones showed the correct insert size and orientation by R-DNA analysis. One of these clones was designated mpl8/SacI/TPA UPA (BC).
B) Kloniranje pBR4a SacI fragmenta v M13mpl8 pBR4a (glej primer 5) SacI fragmenta kloniramo vB) Cloning of the pBR4a SacI fragment into M13mpl8 pBR4a (see example 5) The SacI fragment is cloned into
M13mpl8. Eden izmed klonov ima pravilno velikost inserta in A R orientacijo in ga navajamo kot mp18/SacI/TPA UPA (BR).M13mpl8. One of the clones has the correct insert size and A R orientation and is referred to as mp18/SacI/TPA UPA (BR).
C) Delecijska mutageneza na TPA-UPA hibrid—konstruktihC) Deletion mutagenesis on TPA-UPA hybrid constructs
1) Konstrukcija K2UPAB (BC) /npr. tPA(1-3)-tPA(176-275)uPA(159-411)/1) Construction K2 UPAB (BC) /e.g. tPA(1-3)-tPA(176-275)uPA(159-411)/
Delečijsko mutagenezo izvedemo tako, kot je opisano v splošnem protokolu (glej primer 14) na mpl8/SacI/Deletion mutagenesis was performed as described in the general protocol (see Example 14) on mpl8/SacI/
A BA B
TPA UPA (BC). Pozitivne klone, dobljene pri hibridizaciji, potrdimo z restrikcijsko analizo s SacI. Pri mutantih opazimo ^ 1380 bp trak, v primeri z divjim tipom,ki ‘daje ~-d900 bp fragment. Mutanti so nadalje potrjeni s prebavnim EcoRI.TPA UPA (BC). Positive clones obtained from hybridization are confirmed by restriction analysis with SacI. In mutants, a ~1380 bp band is observed, in the case of the wild type, which gives a ~900 bp fragment. Mutants are further confirmed by EcoRI digestion.
En mutantni klon s pravilno strukturo navajamo kot mpl8/One mutant clone with the correct structure is referred to as mpl8/
BB
SacI/K2UPA (BC). Izpustitev verificiramo z metodo verižnoterminatorskega sekvenciranja ob uporabi sekvencirnega primerja s sekvencoSacI/K2 UPA (BC). The deletion is verified by chain terminator sequencing using a sequencing primer with the sequence
5’ CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGT-G 3’5' CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGT-G 3'
Ta primer je komplementaren kodirnemu vlaknu t-PA(853-821) z napačno vezavo v legi 838 (t-PA).This example is complementary to the coding strand t-PA(853-821) with a mispair at position 838 (t-PA).
2) Konstrukcija FUPAB(BC) /npr. tPA(1-49)-tPA(262-275 )uPA(159-411)2) Construction FUPAB (BC) /e.g. tPA(1-49)-tPA(262-275 )uPA(159-411)
Delečijsko mutagenezo izvedemo tako, kot je opisano v A B v splošnem protokolu (glej primer 14), na mpl8/SacI/TPA UPA (BC). Pozitivne klone, dobljene pri hibridizaciji, potrdimo z restrikcijsko analizo s Sacl. Pri mutantih uporabimo ^1200 bp trak v primeri z divjim tipom, ki daje ^1900 bp fragment.Deletion mutagenesis was performed as described in A B in the general protocol (see Example 14) on mpl8/SacI/TPA UPA (BC). Positive clones obtained from hybridization were confirmed by restriction analysis with SacI. For mutants, a ~1200 bp band was used in the wild-type primer, which gave a ~1900 bp fragment.
Mutanti so nadalje potrjeni s prebavnim EcoRI. En mutanten B klon s pravilno strukturo navajamo kot mpl8/SacI/FUPA (BC). Delecijo verificiramo z metodo verižno-terminatorskega sekvenciranja ob uporabi sekvencirnega primerja s sekvencoThe mutants were further confirmed by EcoRI digestion. One mutant B clone with the correct structure is designated mpl8/SacI/FUPA (BC). The deletion was verified by chain-terminator sequencing using a sequencing primer with the sequence
S’ CAGAGCCCCCCCGGTGC 3’·S’ CAGAGCCCCCCCGGTGC 3’·
Ta primer je komplementaren kodirnemu vlaknu' u-PA(666-682).This primer is complementary to the coding strand' of u-PA(666-682).
3) Konstrukcija FK2UPAB(.BC) (npr. tPA( 1-49)-tPA( 176-275)uPA(159-411)3) Construction of FK2 UPAB (.BC) (e.g. tPA( 1-49)-tPA( 176-275)uPA(159-411)
Delečijsko mutagenezo izvedemo, kot je opisano v splošnem protokolu (glej primer 14) na mpl8/SacI/TPAAUPAB(BC). Pozitivni kloni, dobljeni pri hibridizaciji, so potr- 94 jeni z restrikcijsko analizo s Sacl. Pri mutantih opazimo /v1470 bp trak v primeri z divjim tipom, ki daje <^1900 bp fragment. Mutante nadalje potrdimo s prebavnim EcoRI. En mutantni klon s pravilno strukturo navajamo kot mpl8/SacI/ KF2UPA (BC). Delecijo verificiramo z metodo verižno-terminatorskega sekvenciranja z uporabo sekvencirnega primerj s sekvencoDeletion mutagenesis was performed as described in the general protocol (see Example 14) on mpl8/SacI/TPAA UPAB (BC). Positive clones obtained from hybridization were confirmed by restriction analysis with SacI. In mutants, a band at 1470 bp in the wild-type primer was observed, which yielded a <^1900 bp fragment. Mutants were further confirmed by EcoRI digestion. One mutant clone with the correct structure was designated mpl8/SacI/ KF2 UPA (BC). The deletion was verified by chain-terminator sequencing using a sequencing primer with the sequence
5’ CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGTG 3’.5’ CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGTG 3’.
Primer je komplementaren h kodirnemu vlaknu t-PA(853-821) z napačno vezavo v legi 838 (t-PA).The primer is complementary to the coding strand t-PA(853-821) with a mismatch at position 838 (t-PA).
4. Konstrukcija FGK2UPAB(BC) /npr. tPA(1-86)-tPA(176-275)uPA(159-411).4. Construction of FGK2 UPAB (BC) /e.g. tPA(1-86)-tPA(176-275)uPA(159-411).
Delecijsko mutagenezo izvedemo tako, kot je opisano v splošnem protokolu (glej primer 14) na mpl8/SacI/Deletion mutagenesis was performed as described in the general protocol (see Example 14) on mpl8/SacI/
A BA B
TPA UPA (BC). Pozitivnizkloni, dobljeni pri hibridizaciji, so potrjeni z restrikcijsko analizo s Sacl. Pri mutantih opazimo ^1580 bp trak v primeri z divjim tipom, ki daje m 1900 bp fragment. Mutante nadalje potrdimo s prebavnim EcoRI. En mutantni klon s pravilno strukturo navajamo kot mp18/SacI/FGK2UPA (BC). Delecijo verificiramo z metodo verižno-terminatorskega sekvenciranja ob uporabi sekvencir nega primerja s sekvencoTPA UPA (BC). Positive clones obtained from hybridization are confirmed by restriction analysis with SacI. In mutants, a ~1580 bp band is observed in the wild-type primer, which gives a ~1900 bp fragment. Mutants are further confirmed by EcoRI digestion. One mutant clone with the correct structure is designated mp18/SacI/FGK2 UPA (BC). The deletion is verified by chain-terminator sequencing using a sequencing primer with the sequence
5’ CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGTG 3\5' CCCAGTGCCTGGGCATTGGGGTTCTGTGCTGTG 3\
Ta primer je komplementaren kodirnemu'vlaknu t-PA(853-821 ) z napačno vezavo v legi 838 (t-PA).This example is complementary to the t-PA(853-821) coding strand with a mispair at position 838 (t-PA).
5) Podobne delecijske protokole mutageneze uporabimo za ustvarjanje5) Similar deletion mutagenesis protocols are used to create
K2UPAB(BR) [tPA(l-3)-tPA(176-262)-uPA(132-411)]K2 UPAB (BR) [tPA(1-3)-tPA(176-262)-uPA(132-411)]
FUPAB(BR) [tPA(1-4 9)-uPA(134-411)]FUPAB (BR) [tPA(1-49)-uPA(134-411)]
FK2UPAB(BR) [tPA(l-49)-tPA(176-262)-uPA(132-411)] in FGK2UPAB(BR) [tPA(1-86)-tPA(176-262)-uPA(132-411)].FK2 UPAB (BR) [tPA(1-49)-tPA(176-262)-uPA(132-411)] and FGK2 UPAB (BR) [tPA(1-86)-tPA(176-262)-uPA(132-411)].
D) Uvedba hibridnih PA kodirnih sekvenc v ekspresijskivvek-tor celice sesalcevD) Introduction of hybrid PA coding sequences into a mammalian cell expression vector
1. Uvedba FUPAB(BC), K2UPAB(BC), FK2UPAB(BC) in FGK2UPAB(BC).1. Introduction of FUPAB (BC), K2UPAB (BC), FK2UPAB (BC) and FGK2 UPAB (BC).
RF DNA iz mpl8/SacI/KaUPAB(BC), mpl8/SacI7FUPAB(BC), mp.l8/SacI/FKaUPAB(BC) in mpl8/SacI/TGK2UPAB(BC) vsakokrat . s razrežemo s SacI. Manjšega izmed obeh nastalih fragmentov izoliramo in vežemo na SacI razrezan pBR4a (glej primer 5) vektorski fragment, preveden v E.coli HB101 ter pravilno orientacijo in pravilno velikost insertov potrdimo s prebavljanjem z BamHI oz.SacI. Nastale plazmide označimo kot pCGC5/K2UPA , pCGC6/FUPAB, pCGC7/FK2UPABoz. pCGC8/FGKaUPAB.RF DNA from mpl8/SacI/Ka UPAB (BC), mpl8/SacI7FUPAB (BC), mp.l8/SacI/FKaUPAB (BC) and mpl8/SacI/TGK2UPAB (BC) were each . s digested with SacI. The smaller of the two resulting fragments was isolated and ligated to the SacI digested pBR4a (see example 5) vector fragment, translated into E.coli HB101, and the correct orientation and correct size of the inserts were confirmed by digestion with BamHI or SacI. The resulting plasmids were designated as pCGC5/K2UPA , pCGC6/FUPAB , pCGC7/FK2UPAB or pCGC8/FGKa UPAB .
2. Podobno 'K2UPAB(BR), FUPAB(BR), FK2UPAB(BR) in FGK2UPAB(BR) DNA (glej zgoraj) vsakokrat uvedemo v pBR4a. Dobljene plazmide označimo kot pBR5, pBR6, pBR7 oz.pBR8.2. Similarly, 'K2 UPAB (BR), FUPAB (BR), FK2UPAB (BR) and FGK2UPAB (BR) DNA (see above) are each introduced into pBR4a. The resulting plasmids are designated as pBR5, pBR6, pBR7 and pBR8, respectively.
PRIMER 19: Ekspresijski vektorji sesalcev, ki obsegajo DHFR genEXAMPLE 19: Mammalian expression vectors comprising the DHFR gene
Plazmid pSV2dhfr (ATCC 37145) je plazmid, ki dopušča selekcijo transformantov DHFR-vsebujočih celic s selekcijo ob uporabi anfifolatnega zdravila methotrexata ali selekcijo DHFR+ transformantov DHFR CHO celic /DUKXBI celice; G.Urlaub,Plasmid pSV2dhfr (ATCC 37145) is a plasmid that allows the selection of DHFR-containing cell transformants by selection using the amphifolate drug methotrexate or the selection of DHFR+ transformants of DHFR CHO cells/DUKXBI cells; G.Urlaub,
- 96 Proč. Natl. Acad. Sci. U.S.A. 77, 4216-4220 ( 1980)/. Na eno samo BamHI mesto tega plazmida se da klonirati BamHI fragment pCGA28, ki vsebuje modularni t-PA gen. Plazmide, ki vsebujejo katerokoli izmed obeh možnih orientacij, označimo kot pCGA700a/tPA in pCGA7,00b/tPA. Obe lahko uporabimo za ekspresijo t-PA v celicah tkivne kulture, vendar je prednostna pCGA700a/tPA, kjer je transkripcija t-PA gena v isti smeri < kot transkripcija DHFR gena, ker ta orientacija pogosto vodi do rahlo višjih ekspresijskih nivojev, kot pa s plazmidi, ki se konvergentno transkribirajo. (prepisujejo).- 96 Proc. Natl. Acad. Sci. U.S.A. 77, 4216-4220 (1980)/. Into a single BamHI site of this plasmid, the BamHI fragment of pCGA28, which contains the modular t-PA gene, can be cloned. Plasmids containing either of the two possible orientations are designated pCGA700a/tPA and pCGA7,00b/tPA. Both can be used for expression of t-PA in tissue culture cells, but pCGA700a/tPA is preferred, where transcription of the t-PA gene is in the same direction as transcription of the DHFR gene, because this orientation often leads to slightly higher expression levels than with plasmids that are convergently transcribed. (transcribe).
_ Na analogen način lahko modularne gene, ki kodirajo za hibridne plazminogene aktivatorje (spodaj) iz plazmidov pBR1a/tPA, pBR2a/UPAATPAB, pCGC1/UPAATPAB, in pCGC2/UK2TPABkombiniramo kot BamHI fragmente z DHFR genom pCGA700a/tPA, da se tvorijo plazmidi pGGA701a/tPA, pCGA702a/UPAATPAB, pCGA705a/ UPAATPAB oz. pCGA707a/UK2T PAB, kjer se modularni plazminogenski aktivatorski gen transkribira v isti smeri kot DHFR gen, ter pCGA701b/tPA, pCGA702b/UPAATPAB, pCGA705b/UPAATPAB, g_ In an analogous manner, the modular genes encoding hybrid plasminogen activators (below) from plasmids pBR1a/tPA, pBR2a/UPAA TPAB , pCGC1/UPAA TPAB , and pCGC2/UK2TPAB can be combined as BamHI fragments with the DHFR gene of pCGA700a/tPA to form plasmids pGGA701a/tPA, pCGA702a/UPAA TPAB , pCGA705a/UPAA TPAB , and pCGA707a/UK2T PAB , respectively, where the modular plasminogen activator gene is transcribed in the same direction as the DHFR gene, and pCGA701b/tPA, pCGA702b/UPAA TPAB , pCGA705b/UPAA TPAB , g
j£GA707b/UK2TPA , kjer se oba gena transkribirata v nasprotnih smereh. Zaradi prisotnosti BamHI sekvence v deležu, ki kodira za u-PA B-verige, se da modularni plazminogenski aktivatorskij£GA707b/UK2 TPA , where both genes are transcribed in opposite directions. Due to the presence of a BamHI sequence in the region coding for the u-PA B-chain, a modular plasminogen activator gene is possible.
D gen izolirati samo z delnim rezom (2 izmed 3 BamHI leg) neo plazmida, čemu sledi izolacija primernega fragmenta (glej slike) z agarozno gelno elektroforezo.The D gene can be isolated only by partial excision (2 of the 3 BamHI legs) of the neo plasmid, followed by isolation of the appropriate fragment (see pictures) by agarose gel electrophoresis.
Tako izPBR3a/uPA, pBRAa/TPA^PA5, pBR5/K2UPAB, pBR6/FUPAB, pBR7/FK2UPAB, pBR8/FGK2UPAB, pCGC3/UK2UPAB, pCGC4a/TPAAUPAB, pCGC5/K2UPAB, pCGC6/FUPAB, pCGC7/FK2UPAB, in pCGC8/FGK2UPAB lahko konstruiramo pCGA7O3a/uPA, pCGA/OAa/TPA^PA , pCGA7O5a/K2UPAB, pCGA7O8a/FUPAB, pCGA706a/FK2UPAB,PCGA7O7a/FGK2UPAB, pCGA709a/UK2UPAB, pCGA711a/TPAAUPAB, pCGA712a/K2UPAB, pCGA713a/FUPAB, pCGA714a/FK2UPAB oz.Thus, fromP BR3a/uPA, pBRAa/TPA^PA5 , pBR5/K2UPAB , pBR6/FUPAB , pBR7/FK2UPAB , pBR8/FGK2UPAB , pCGC3/UK2UPAB , pCGC4a/TPAA UPAB , pCGC5/K2UPAB , pCGC6/FUPAB , pCGC7/FK2UPAB , and pCGC8/FGK2UPAB we can construct pCGA7O3a/uPA, pCGA/OAa/TPA^PA , pCGA7O5a/K2UPAB , pCGA7O8a/FUPAB , pCGA706a/FK2 UPAB ,P CGA7O7a/FGK2 UPAB , pCGA709a/UK2UPAB , pCGA711a/TPAA UPAB , pCGA712a/K2UPAB , pCGA713a/FUPAB , pCGA714a/FK2 UPAB or
pCGA715a/FGK2UPAB, kjer se vsi plazminogeni aktivatorski geni transkribirajo (prepišejo) v isti smeri kot DHFR gen in nadalje pCGA7O3b/uPA, pCGA7O4b/TPAAUPAB, pCGA708b/FUPAB, pCGA705b/K2UPAB, pCGA706b/FK2UPAB, pCGA707b/FGK2UPAB, pCGA709b/UK2UPAB, pCGA711b/TPAAUPAB, pCGA712b/K2UPAB, pCGA713b/FUPAB, pCGA714b/FK2UPAB in. pCGA715b/FGK2UPAB, kjer--se?:Oba genanekonvergentno transkribirata.pCGA715a/FGK2 UPAB , where all plasminogen activator genes are transcribed in the same direction as the DHFR gene and further pCGA7O3b/uPA, pCGA7O4b/TPAA UPAB , pCGA708b/FUPAB , pCGA705b/K2UPAB , pCGA706b/FK2UPAB , pCGA707b/FGK2 UPAB , pCGA709b/UK2 UPAB , pCGA711b/TPAA UPAB , pCGA712b/K2 UPAB , pCGA713b/FUPAB , pCGA714b/FK2UPAB and. pCGA715b/FGK2UPAB , where--are?: Both genes non-convergently transcribed.
PRIMER 20: Priprava hibridnih plazmiriogenskih aktivatorjev s transformiranimi celicami sesalcevEXAMPLE 20: Preparation of hybrid plasminogen activators with transformed mammalian cells
A) Vzdrževanje in DNA transiekcija celic tkivnih kultur; splošni postopekA) Maintenance and DNA transfection of tissue culture cells; general procedure
DNA konstrukte izrazimo v DUKXBI, mutantu ovarijskih celic kitajskega hrčka (CHO), ki nimajo encima dihidrofolatreduktaze /G. Urlaub et al., Proč. Nat.Acad. Sci USA 77, 4216-4220 (1980)/.DUKXB1 celice gojimo v alfa-MEM mediju, ki vsebuje nukleozide (GIBCO), katerim smo dodali 5% fetusnega telečjega seruma.The DNA constructs were expressed in DUKXBI, a mutant of Chinese hamster ovary (CHO) cells lacking the enzyme dihydrofolate reductase /G. Urlaub et al., Proc. Nat.Acad. Sci USA 77, 4216-4220 (1980)/. DUKXB1 cells were grown in alpha-MEM medium containing nucleosides (GIBCO) supplemented with 5% fetal calf serum.
Celice 'posadimo' z gostoto 10 000/cm v 6-utorne multiplošče (3,4 cm premera) in transformiramo s 4 ^ig DNA: DNA raztopimo v količini 50 ^ig/ml v 10 mM Tris/HCl pH 7,0, ki vsebuje 0,1 mM EDTA, hlajen na ledu v teku 5 minut, dodamo 0,25 volumnov 1 M CaCl2 in inkubiramo za 10 minut na ledu. Zmes nato pomešamo z enakim volumnom 2 x HBS (50mM Hepes,Cells were seeded at a density of 10,000/cm in 6-well multiplates (3.4 cm diameter) and transformed with 4 µg of DNA: DNA was dissolved at 50 µg/ml in 10 mM Tris/HCl pH 7.0 containing 0.1 mM EDTA, cooled on ice for 5 minutes, 0.25 volumes of 1 M CaCl2 were added and incubated for 10 minutes on ice. The mixture was then mixed with an equal volume of 2 x HBS (50 mM Hepes,
280 mM NaCl, 0,75 mM Na2HPO4, 0,75 mM NaH2P04, pH 7,12, čemur sledi še ena 10-minutna inkubacija na ledu. Končno to skupno oborino DNA-Ca-fosfata dodamo h gojilnemu mediju in inkubiramo celice z DNA za 16—18 ur, čemur sledi glicerolni280 mM NaCl, 0.75 mM Na2 HPO4 , 0.75 mM NaH2 P04 , pH 7.12, followed by another 10-minute incubation on ice. Finally, this total DNA-Ca-phosphate precipitate is added to the culture medium and the cells are incubated with DNA for 16-18 hours, followed by glycerol
- 98 šo^, t.j. celice splaknemo s TBS (80 g/1 NaCl, 3,8 g/1 KC1, g/1 Na2HPO4.2H20, 0,114 g/1 CaCl2.2H20, 0,11 g/1 MgCl2· 6H20, 25 mM Tris/HCl pH 7,5), inkubiramo za 1 minuto z 20% (vol/vol) glicerola v TBS, ponovno splaknemo s TBS in gojimo 24 ur v mediju tkivne kulture. Celice nato tripsiniziramo in jih prenesemo v petrijevke s premerom 8 cm. Naslednji dan nadomestimo prvotni gojilni medij brez selektivnega sredstva z medijem, ki vsebuje 1 mg/ml geneticina. Medij nadomeščamo vsak tretji ali četrti dan. Kolonije lahko vidimo okoli 14. dne. Celice iz individualnih kolonij izoliramo z odpraskanjem s konico pipete, pri čemer jih istočasno vsrkamo v konico, polnjeno z raztopino tripsina, ter vsako prenesemo v utor 24utorne multiplošČe, v katero dovajamo medij, ki vsebuje geneticin. Ko so konfluentne, cepimo te kulture na utore 6-utorne multiplošČe in zatem v petrijevke s premerom 8 cm.- 98 scho^, i.e. the cells are rinsed with TBS (80 g/l NaCl, 3.8 g/l KC1, g/l Na2 HPO4 .2H2 0, 0.114 g/l CaCl2 .2H2 0, 0.11 g/l MgCl2 · 6H2 0, 25 mM Tris/HCl pH 7.5), incubated for 1 minute with 20% (vol/vol) glycerol in TBS, rinsed again with TBS and grown for 24 hours in tissue culture medium. The cells are then trypsinized and transferred to 8 cm diameter Petri dishes. The next day, the original culture medium without selective agent is replaced with medium containing 1 mg/ml geneticin. The medium is replaced every third or fourth day. Colonies can be seen around the 14th day. Cells from individual colonies are isolated by scraping with a pipette tip, simultaneously aspirating them into a tip filled with trypsin solution, and each is transferred to a well of a 24-well multiplate, into which medium containing geneticin is added. When confluent, these cultures are plated onto wells of a 6-well multiplate and then into 8 cm diameter Petri dishes.
B. Poskusi na agarozni plošči za plazminogenske aktivatorjeB. Agarose plate assays for plasminogen activators
Ti občutljivi poskusi za plazminogenske aktivatorje uporabljajo agarozne gele, katerim je dodan plazminogen (izhodna raztopina je pripravljena z raztapljanjem plazminogena Sigma A-6877 v količini 1 mg/ml ter z dvakratnim dializiranjem proti 100 volumnom 50 mM Tris/HCl pH 8,0), ali pa kazein (dodan kot nemastno mleko)ali fibrin (dodan kot fibrinogen plus trombin). Vzorec, ki vsebuje plazminogenski aktivator, vnesemo v luknjice, izdolbljene v 4 mm debel sloj agaroze^, ter gel zatem inkubiramo pri 37°C. Nato ugotavljamo encimatsko aktivnost s tem, da plazminogenski aktivator radialno difundira od utora z vzorcem, pretvarja plazminogen v gelu v plazmin, kateri prebavi kazein ali fibrin, s čemer tvori jasen obroč v neprozornem gelu okoli utora z vzorcem. Radij obroča, merjen od roba utora z vzorcem, je merilo za količino aktiviranega plazminogena. Poskus ne kaže linearnega odziva na količino dodanega plazminogenskega aktivatorja. Za preizkus manjših količin plazminogenskega aktivatorja lahko inkubacijo podaljšamo na več dni. Način dela in kalibriranje kazeinskega poskusa sta taka, kot so opisali Tang et al.These sensitive plasminogen activator assays use agarose gels spiked with plasminogen (the stock solution is prepared by dissolving Sigma A-6877 plasminogen at 1 mg/ml and dialyzing twice against 100 volumes of 50 mM Tris/HCl pH 8.0), or casein (added as nonfat milk) or fibrin (added as fibrinogen plus thrombin). The sample containing the plasminogen activator is added to wells drilled into a 4 mm thick agarose layer^ and the gel is then incubated at 37°C. Enzymatic activity is then determined by the plasminogen activator diffusing radially from the sample well, converting the plasminogen in the gel to plasmin, which digests the casein or fibrin, forming a clear ring in the opaque gel around the sample well. The radius of the ring, measured from the edge of the sample well, is a measure of the amount of activated plasminogen. The assay does not show a linear response to the amount of plasminogen activator added. To test smaller amounts of plasminogen activator, the incubation can be extended to several days. The procedure and calibration of the casein assay are as described by Tang et al.
/Ann. Ν.Υ. Acad. Sci. 434, 536-540 (1984)/, le da uporabimo namesto 2% (mas/vol) Carnation nemastnega mlečnega prahu 12,5% (vol/vol) steriliziranega (UHT) nemastnega mleka Migros Corp. (Švica). Če uporabimo fibrin /Granelli-Piperno in Reich, J. Exp. Med. 148, 223-234 (1978)/ kot substrat, raztopimo 0,2 g agaroze v 15 ml 0,9% NaCl in ohladimo na 42°C Pri tej točki dodamo 5 ml 0,9% NaCl, ki vsebuje 80 mg govejega fibrinogena (Sigma F-8630), 0,1 ml plazminogenske raztopine (zgoraj) in 0,1 ml 100 mg/ml natrijevega azida pri 42°C. Končno dodamo 0,2 ml govejega trombina (Sigma T-6634), raztopljenega v količini 16,6 NIH enot/ml v 0,9%-nem NaCl), nakar zmes hitro izlijemo v petrijevke (s premerom 8 cm) in pustimo ohladiti na sobno temperaturo v 1 uri. Nastali gel je debel okoli 4 mm in ga lahko skladiščimo pri 4°C nekaj dni, ali pa ga takoj uporabimo na enak način kot zgornji gel, ki vsebuje kazein./Ann. Ν.Υ. Acad. Sci. 434, 536-540 (1984)/, except that instead of 2% (w/v) Carnation nonfat milk powder, 12.5% (v/v) sterilized (UHT) nonfat milk from Migros Corp. (Switzerland) is used. If fibrin is used /Granelli-Piperno and Reich, J. Exp. Med. 148, 223-234 (1978)/ as a substrate, dissolve 0.2 g of agarose in 15 ml of 0.9% NaCl and cool to 42°C. At this point, add 5 ml of 0.9% NaCl containing 80 mg of bovine fibrinogen (Sigma F-8630), 0.1 ml of plasminogen solution (above), and 0.1 ml of 100 mg/ml sodium azide at 42°C. Finally, 0.2 ml of bovine thrombin (Sigma T-6634) dissolved at 16.6 NIH units/ml in 0.9% NaCl was added, and the mixture was quickly poured into Petri dishes (8 cm in diameter) and allowed to cool to room temperature within 1 hour. The resulting gel was about 4 mm thick and could be stored at 4°C for several days, or used immediately in the same way as the casein gel above.
- ίσο C. Priprava hibridnih PA proteinov v celicah hrčka- ίσο C. Preparation of hybrid PA proteins in hamster cells
CHO DUKXB1 celice transformiramo z DNA plazmidov pBR1A, pBR1B, pBR2A, pBR2B, pBR3'A, pBR3B, pBR5, pBR7, pBR8, pCGC1 , pCGC2, pCGC3, pCGC4a, pCGČ5, pCGC6, pCGC7 oz. pCGC8, kot je zgoraj opisano (primer 20A). Kolonije se pojavijo okoli 10-ega dne, poberemo pa jih okoli 15-ega dne tako, kot je zgoraj opisano, in 2 tedna kasneje se število celic dovolj poveča, da lahko izmerimo PA kot je zgoraj opisano. Netransformirane celice in celične linije, transformirane s pBR1B, pB:R2B, pBR3B, ki vsebujejo vključeni SacI fragment v nai sprotni orientaciji, ne proizvedejo ugotovljivih količin PA.CHO DUKXB1 cells were transformed with DNA from plasmids pBR1A, pBR1B, pBR2A, pBR2B, pBR3'A, pBR3B, pBR5, pBR7, pBR8, pCGC1 , pCGC2, pCGC3, pCGC4a, pCGČ5, pCGC6, pCGC7, and pCGC8, respectively, as described above (Example 20A). Colonies appeared around day 10, were picked around day 15 as described above, and 2 weeks later the cell number increased sufficiently to allow PA to be measured as described above. Untransformed cells and cell lines transformed with pBR1B, pB:R2B, pBR3B containing the included SacI fragment in the upstream orientation did not produce detectable amounts of PA.
D. Encimska aktivnost v medijih, kondicioniranih s transformiranimi CHO celicamiD. Enzyme activity in media conditioned by transformed CHO cells
Kondicioniran medij iz celic, transformiranih s plazmidom,in kontrolnih CHO celic, pripravimo z gojenjem 200,000-500,000 celic/ml v teku 24 ur v Alpha-MEM, z nukleozidi ter 5%-nim fetusnim telečjim serumom in 0,03 ml inkubiramo na agaroznih ploščah, ki vsebujejo kazein ali fibrin, v spodaj navedenem časovnem obdobju. Na fibrinski plošči ugotovimo minimalno aktivnost ozadja, domnevno zaradi endogenega t-PA hrčka, v mediju, kondicioniranem z DUKXB1. Na kazeinskih ploščah se ne pojavlja obroč, če vzorce hibridnega proteina pomešamo s 3 μΐ kunčjih anti-tPA protiteles (vzgojenih proti očiščenim Bowes melanomskim t-PA) ali z anti-urokinaznimi protitelesi (vzgojenimi proti Serono urokinazi).Conditioned media from plasmid-transformed and control CHO cells were prepared by culturing 200,000-500,000 cells/ml for 24 hours in Alpha-MEM supplemented with nucleosides and 5% fetal calf serum and incubating 0.03 ml on agarose plates containing casein or fibrin for the time periods indicated below. Minimal background activity was observed on the fibrin plate, presumably due to endogenous hamster t-PA, in the DUKXB1-conditioned medium. No ring was observed on the casein plates when hybrid protein samples were mixed with 3 μΐ rabbit anti-tPA antibodies (raised against purified Bowes melanoma t-PA) or anti-urokinase antibodies (raised against Serono urokinase).
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Anti-tPA protitelo ne inhibira u-PA encima, niti anti-urokinazn^ protitelo ne inhibira t-PA v signifikantni meri. Rezultati so zbrani v tabeli 1.The anti-tPA antibody does not inhibit the u-PA enzyme, nor does the anti-urokinase antibody inhibit t-PA to a significant extent. The results are summarized in Table 1.
Tabela 1: Učinkovitost različnih plazminogenskih aktivatorjev transformirni plazmid premer obročaTable 1: Efficacy of different plasminogen activators transforming plasmid ring diameter
PRIMER 21: Priprava hibridomskih celic in izolacija monoklonalnih protitelesEXAMPLE 21: Preparation of hybridoma cells and isolation of monoclonal antibodies
a) Vir imunogena: Vzorec pol-očiščenega naravnega humanega melanoma t-PA) z ocenjeno čistočo nad 90%.a) Source of immunogen: Semi-purified natural human melanoma sample (t-PA) with an estimated purity of over 90%.
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b) Imunizacijski protokol: Tri skupine BALB/c miši (Tierfarm Sisseln, Švica), stare 10 do 14 tednov, imuniziramo z injekciv oba zadnja podplata ter subkutano s 100 ^ug melanoma t-PA, emulgiranega v popolnem Freundovem adjuvantu (Difco). Zatem dobi prva skupina (št. 405) 10/Ug t-PA v nepopolnem adjuvantu vsak teden v teku 6 tednov, medtem ko druga skupina (406) dobi isto količino na vsaka 2 tedna. Tretji skupini (407) damo dvakrat 50 ^ug t-PA v intervalih po 3 tedne.b) Immunization protocol: Three groups of BALB/c mice (Tierfarm Sisseln, Switzerland), aged 10 to 14 weeks, were immunized by injection into both hind paws and subcutaneously with 100 µg of melanoma t-PA emulsified in complete Freund's adjuvant (Difco). The first group (no. 405) then received 10 µg of t-PA in incomplete adjuvant every week for 6 weeks, while the second group (no. 406) received the same amount every 2 weeks. The third group (no. 407) was given 50 µg of t-PA twice at 3-week intervals.
Vsem živalim vzamemo kri četrti in osmi teden. Za zadnjo injekcijo damo 100 ^ug t-PA v PBS i.p. in 4 dni kasneje izvedemo fuzijo celic vranice s SP2/o mielorasko linijo, v skladu s standardnim postopkom. Za fuzijo uporabimo samo tiste miši, ki imajo visok anti-t-PA protitelesni titer.All animals were bled at weeks 4 and 8. For the final injection, 100 µg t-PA in PBS was administered i.p. and 4 days later, spleen cells were fused with the SP2/o myeloid cell line according to standard procedures. Only mice with high anti-t-PA antibody titers were used for fusion.
c) Celična fuzija: Vse fuzijske eksperimente izvedemo v skladu s postopkom po G. Kohler in C. Milstein /Naturec) Cell fusion: All fusion experiments were performed according to the procedure of G. Kohler and C. Milstein /Nature
256, 495 (1975) ob uporabi neizločevalne Sp 2/0-Agl4 mielomske vrste /M. Shulman, C.D. Wilde in G. Kohler, • 8256, 495 (1975) using the non-secreting Sp 2/0-Agl4 myeloma line /M. Shulman, C.D. Wilde and G. Kohler, • 8
Nature 276, 269 (1978)/. 10 vraničnih celic pomešamo z mielomskih celic v prisotnosti 1 ml 50 % polietilenglikola (PEG 1500, Serva). Po izpiranju celice resuspendiramo v 48 ml standardnega Dulbecco minimalnega esencialnega medija (Gibco No. 0422501). 3χ10θ normalnih mišjih peritonealnih eksudatnih celic na fuzijo dodamo kot hranilne celice. Celice porazdelimo v 48x1 ml costar utore in jih hranimo trikrat tedensko s standardnim HAT selekcijskim medijem v teku 3 do 6 tednov. Ko postane rast hibridomskih celicNature 276, 269 (1978)/. 10 spleen cells were mixed with myeloma cells in the presence of 1 ml of 50% polyethylene glycol (PEG 1500, Serva). After washing, the cells were resuspended in 48 ml of standard Dulbecco's minimal essential medium (Gibco No. 0422501). 3χ10θ normal mouse peritoneal exudate cells per fusion were added as feeder cells. The cells were distributed into 48x1 ml costar wells and fed three times a week with standard HAT selection medium for 3 to 6 weeks. When the growth of the hybridoma cells became
103 vidna, izvedemo , skrining supernatantov tako s poskusom direktne antigenske vezave (ELISA) in nevtralizacijo (kazein) (glej spodaj). Rezultati 4 fuzijskih poskusov so naslednji: izmed 192 cepljenih utorov dobimo 192 hibridomov. Izmed teh jih 24 proizvaja anti-t-PA protitelo. Izmed 24 pozitivnih hibridomov jih 14 kloniramo in izmed dobljenih 574 klonov ugotovimo, da jih 31 stabilno proizvaja anti-t-PA mAB. Tri odznjih (klone 4O5B.33.3, 4O6A.23.7 in 4O7A. 15.27) injiciramo v miši ter proizvedemo ascites tekočine za nadaljnje proučevanje.103 visible, we screened the supernatants by both direct antigen binding assay (ELISA) and neutralization (casein) assays (see below). The results of the 4 fusion experiments are as follows: from 192 grafted wells, 192 hybridomas were obtained. Of these, 24 produced anti-t-PA antibody. Of the 24 positive hybridomas, 14 were cloned and of the 574 clones obtained, 31 were found to stably produce anti-t-PA mAb. Three of these (clones 4O5B.33.3, 4O6A.23.7 and 4O7A. 15.27) were injected into mice and ascites fluid was produced for further study.
d) Izolacija in čiščenje monoklonalnega protitelesa;d) Isolation and purification of monoclonal antibody;
BALB/c miši, stare 8 do 10 tednov, (Tierfarm Sisseln,BALB/c mice, 8 to 10 weeks old, (Tierfarm Sisseln,
Švica), intraperitonealno predobdelarao z 0,3 ml pristana (Aldrich). 2 do 3 tedne kasneje jim intraperitonealno inokuliramo 2 do 5x10^ kloniranih hibridomskih celic 4O5B.33.3, 4O6A.23.7 in 4O7A.15.27 in 0,2 ml pristana. Po 8 do 10 dneh zberemo ascites tekočino, centrifugiramo pri 800xg in shranimo pri -20°C.Switzerland), intraperitoneally pretreated with 0.3 ml pristane (Aldrich). 2 to 3 weeks later, they were inoculated intraperitoneally with 2 to 5x10^ cloned hybridoma cells 4O5B.33.3, 4O6A.23.7 and 4O7A.15.27 and 0.2 ml pristane. After 8 to 10 days, ascites fluid was collected, centrifuged at 800xg and stored at -20°C.
Odledeneno ascites tekočino centrifugiramo pri 50000 x g v teku 60 minut. Sloj maščobe, ki plava na površini, skrbno odstranimo in uravnamo koncentracijo proteina na 10 do 12 mg/ml.The thawed ascites fluid is centrifuged at 50,000 x g for 60 minutes. The fat layer floating on the surface is carefully removed and the protein concentration is adjusted to 10 to 12 mg/ml.
Surovi imunoglobulin oborimo z dokapavanjem 0,9 volumskih ekvivalentov nasičenega amonijevega sulfata pri 0°C, zatem raztopimo v 20 mM Tris-HCl/50 mM NaCl (pH 7,9) in dializiramo proti istemu pufru.. Dobimo imunoglobu104 linsko frakcijo z DEAE D52 celulozno (Whatman) kromatografijo, ob uporabi pufrskega gradientnega sistema 20 mM Tris-HCl/25-400 mM NaCl, pH 7,9. Jmunoglobulin ponovno oborimo z amonijevim sulfatom in raztopimo v PBS v koncentraciji 10 mg/ml.The crude immunoglobulin was precipitated by dropwise addition of 0.9 volume equivalents of saturated ammonium sulfate at 0°C, then dissolved in 20 mM Tris-HCl/50 mM NaCl (pH 7.9) and dialyzed against the same buffer. The immunoglobulin fraction was obtained by DEAE D52 cellulose (Whatman) chromatography, using a buffer gradient system of 20 mM Tris-HCl/25-400 mM NaCl, pH 7.9. The immunoglobulin was reprecipitated with ammonium sulfate and dissolved in PBS at a concentration of 10 mg/ml.
Gelna elektroforeza z natrijevim dodecilsulfatompoliakrilamidom (SDS-PAGE) pokaže stopnjo čistoče nad 95 % za monoklonalna patb^iteiesa...Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) shows a purity level of over 95% for monoclonal patb^iteiesa...
(' 'i(' 'i
e) Določitev razreda in podrazreda monoklonalnih protiteles?e) Determination of the class and subclass of monoclonal antibodies?
Razred in podrazred monoklonalnih ; proizvedenih s kloniranimi hibridomskimi celicami, določimo po znani imunodifuzijski tehniki Ouchterlonyja (agarsko-gelna imunodifuzijska metoda), ob uporabi razredno in podrazredno specifičnih, kunčjih sprt>fc±teales (Bionetics).The class and subclass of monoclonal antibodies produced by cloned hybridoma cells are determined by the well-known immunodiffusion technique of Ouchterlony (agar-gel immunodiffusion method) using class- and subclass-specific rabbit sputum cultures (Bionetics).
ί ·ί ·
Podrazredi mAb so naslednjirThe subclasses of mAbs are as follows:
405B.33.3 : Ti*405B.33.3 : You*
406A.23.7 : rab*·406A.23.7 : use*·
407A.15.27 : 62atr.407A.15.27 : 62atr.
f) Encimski imuno poskus (ELISA); Mikrotiterske plošče prekrijemo z 0,5 ^ug na utor s t-PA pripravkom (čistoča nad 95 %) v 100 ^ul PBS. Prosto vezivno kapaciteto plošče nasitimo s pufrom z 0,2 % želatine v PBS, ki vsebuje vzorce 0,2 % NaN^ \(m/v), pH 7,4 100^ul, ki vsebujejo monoklonalna protitelesa^ 4O5B.33.3, 4O6A.23.7 oz. 4O7A.15.2, inkubiramo v utorih pri 37°C 2 uri. Plošče izperemo s PBS, ki vsebuje 0,05 % Tween 20, nato inkubiramo pri 37°Cf) Enzyme-linked immunosorbent assay (ELISA); Microtiter plates are coated with 0.5 µg per well of t-PA preparation (purity > 95%) in 100 µl PBS. The free binding capacity of the plate is saturated with a buffer with 0.2% gelatin in PBS containing 0.2% NaN^ \(m/v), pH 7.4. 100 µl samples containing monoclonal antibodies^ 4O5B.33.3, 4O6A.23.7 or 4O7A.15.2 are incubated in the wells at 37°C for 2 hours. The plates are washed with PBS containing 0.05% Tween 20, then incubated at 37°C.
105 za 2 uri s kunčjim, anti-mišjim imunoglobulinskim pripravkom konjugiranim s fosfatazo. Fiksirani encim razvijemo z inkubiranjem (37°C, 30 do 60 minut) z raztopino encimskega substrata. p-nitr6fenilfosfata (1 mg/ml v dietanolaminskem pufru, 10 %, ki vsebuje 0,5 mM MgCl2 in 0,02 % (mas/vol NaN^, pH 9,8), ter izmerimo optično gostoto pri 405”hm.105 for 2 hours with a rabbit anti-mouse immunoglobulin preparation conjugated to phosphatase. The fixed enzyme is developed by incubation (37°C, 30 to 60 minutes) with a solution of the enzyme substrate, p-nitrophenylphosphate (1 mg/ml in diethanolamine buffer, 10%, containing 0.5 mMMgCl2 and 0.02% (w/v NaN2, pH 9.8), and the optical density is measured at 405 nm.
Enak poskus ELISA izvedemo tudi z uporabo urokinaze. Nobeno izmed mAb se ne veže na urokinazo. Vsi mAb so t-PA specifični.The same ELISA experiment is also performed using urokinase. None of the mAbs bind to urokinase. All mAbs are t-PA specific.
g) Poskus s kazeinsko lizo (nevtralizacijski test):g) Casein lysis test (neutralization test):
Da bi določili inhibitorski učinek mAbs, najprej t-PA pomešamo z mAbs 4O5B.33.3, 4O6B.23.7 oz. 407A.15.27, ter inkubiramo za 30 do 60 minut pri 4°C, nakar izvedemo običajni poskus kazein/plazminogenski agar (glej primer 20B). Nobeden izmed mAb ne inhibira t-PA aktivnosti, le da mAb 4O5B.33-3 povzroči zakasnitev (več kot 6 ur) pri kazeinski liži.To determine the inhibitory effect of the mAbs, t-PA was first mixed with mAbs 4O5B.33.3, 4O6B.23.7, and 407A.15.27, respectively, and incubated for 30 to 60 minutes at 4°C, followed by a standard casein/plasminogen agar assay (see Example 20B). None of the mAbs inhibited t-PA activity, except that mAb 4O5B.33-3 caused a delay (more than 6 hours) in casein lysis.
PRIMER 22:EXAMPLE 22:
Čiščenje hibridnega plazminogenskega aktivatorjaja splošen postopekPurification of hybrid plasminogen activator is a general procedure
Ekstrakte iz transformiranih kvasnih celic pripravimo tako, kot je opisano v primeru 16. Ekstrakte iz celic sesalcev, transformiranih s plazmidom, kot CHO celic, pripravimo kot sledi:Extracts from transformed yeast cells are prepared as described in Example 16. Extracts from plasmid-transformed mammalian cells, such as CHO cells, are prepared as follows:
Celice najprej vzgojimo do 70 do 80 %-ne konfluence. Nato celični monosloj splaknemo z medijem,The cells are first grown to 70 to 80% confluence. Then the cell monolayer is rinsed with medium,
106 kot je zgoraj opisano, le da izpustimo serum, čemur sledi gojenje celic v dodatnem obdobju 5 do 7 dni. Medij pospravljamo vsakih 24 ur, istočasno pa dovajamo k celicam svež medij. Tako dobljeni kondicionirani medij nato centrifugiramo pri 5000 x g v teku 30 minut ter filtriramo skozi O,45/Um filter, da odstranimo nezaželene drobce celic, pred afiniteto;' kromatografijo. Kot afinitetno matrico uporabimo bodisi imobilizirani proteazni inhibitor DE-3 iz Erythrina latissima ali imobilizirana protitelesa! na u-PA ali t-PA.106 as described above, except that serum is omitted, followed by culturing the cells for an additional period of 5 to 7 days. The medium is harvested every 24 hours, while fresh medium is added to the cells. The resulting conditioned medium is then centrifuged at 5000 x g for 30 minutes and filtered through a 0.45/Um filter to remove unwanted cell debris, prior to affinity chromatography. Either immobilized protease inhibitor DE-3 from Erythrina latissima or immobilized antibodies to u-PA or t-PA are used as the affinity matrix.
Iz hibridnih PA, ki vsebujejo katalitsko B-verigo t-PA, očistimo kondicionirani medij, pripravljen tako, kot je zgoraj opisano, ali ekstrakte kvasnih celic, ob uporabi protokola, originalno razvitega za čiščenje medija, kondicloniranega z melanomskimi celicami, iz t-PA (glej C.Heussen et al., J. Biol. Chem. 259, 1 1635-1 1638 (1984).Hybrid PAs containing the catalytic B-chain of t-PA are purified from conditioned media prepared as described above or from yeast cell extracts using a protocol originally developed for purifying melanoma cell-conditioned media from t-PA (see C. Heussen et al., J. Biol. Chem. 259, 1 1635-1 1638 (1984).
Vse hibridne PA očistimo ob uporabi poliklonalnih protiteles, vzgojenih v kuncih ali kozah, proti parenteralnim u-PA in t-PA encimom, ali ob uporabi monoklonalnih protiteles (mišjega izvora), vzgojenih proti parenteralnim encimom, s pridržkom da le-ti razpoznajo epitop, prisoten v obravnavanem hibridnem PA (glej primer 21). Izbrano protitelo, imobiliziramo na netopni matrici kot Affigelu ali Sepharosi-4B Kondicionirani medij, pripravljen kot je zgoraj opisano, ali kvasni celični ekstrakt» nato nanesemo na kolono afinitetne matrice, nezaželene proteine pa izperemo z uporabo primernega pufra, npr. Dulbecco’s PBS /0,1 g/1All hybrid PAs are purified using polyclonal antibodies raised in rabbits or goats against the parenteral u-PA and t-PA enzymes, or using monoclonal antibodies (of mouse origin) raised against the parenteral enzymes, provided that they recognize an epitope present in the hybrid PA in question (see Example 21). The selected antibody is immobilized on an insoluble matrix such as Affigel or Sepharose-4B. Conditioned medium prepared as described above, or yeast cell extract, is then applied to the affinity matrix column, and unwanted proteins are eluted using a suitable buffer, e.g. Dulbecco's PBS /0.1 g/l
- ιογ CaCl2, 0,2 g/1 KC1, 0,2 g/1 KH2PO4, 0,047 g/1 MgCl2, 8,0 g/1 NaCl, 1,15 g/1 Na^PO^; J. Exp. Med. 99, 167 (1954)/, nakar eluiramo PA iz kolone ob uporabi kaostropnega sredstva kalijevega tiocianata /prim. M. Einarsson et al., Biochim. Biophys. Acta 830, 1-10 (1985) t ali v šibkem pH pufru kot 0,1-0,2 M glicin-HCl (pH 2,1).- ιογ CaCl2 , 0.2 g/1 KC1, 0.2 g/1 KH2 PO4 , 0.047 g/1 MgCl2 , 8.0 g/1 NaCl, 1.15 g/1 Na^PO^; J. Exp. Med. 99, 167 (1954)/, after which the PA is eluted from the column using the chaotropic agent potassium thiocyanate /cf. M. Einarsson et al., Biochim. Biophys. Acta 830, 1-10 (1985) t or in a weak pH buffer such as 0.1-0.2 M glycine-HCl (pH 2.1).
Po očiščenju ob uporabi monoklonalnih protitelesa imajo hibridne PA čistočo nad 90 %.After purification using monoclonal antibodies, hybrid PAs have a purity of over 90%.
PRIMER 23:EXAMPLE 23:
Čiščenje UK2TPAB(BC)Cleaning UK2 TPAB (BC)
Cr)Cr)
a. Priprava DE-3 Sepharosekolonea. Preparation of DE-3 Sepharosecolones
Na 1 ml s cianogenbnomidom aktivirane SepharosePer 1 ml of cyanogen bromide-activated Sepharose
4B(Pharmacia) pripajamo 5 mg očiščenega inhibitorja iz4B(Pharmacia) is coupled with 5 mg of purified inhibitor from
Erythnina latissima /F.J. Joubert et al., Hoppe-Seyler’Erythnina latissima /F.J. Joubert et al., Hoppe-Seyler'
Zeitschr. Physiol. Chem. 302, 531 ( 1981 ))/, v skladu s proizvajalčevimi navodili. Matrico uravnotežimo z 0,2 M amonijevim acetatnim pufrom s pH 7,0, ki vsebuje 0,2 M (R)Zeitschr. Physiol. Chem. 302, 531 ( 1981 ))/, according to the manufacturer's instructions. The matrix is equilibrated with 0.2 M ammonium acetate buffer, pH 7.0, containing 0.2 M (R)
NaCl, 0,1 % Synperonic in 0,02 % natrijevega azida.NaCl, 0.1% Synperonic and 0.02% sodium azide.
b. Kromatografsko čiščenje UK^TPAB(BC) na DE-3 Sepharose 4^·b. Chromatographic purification of UK^TPAB (BC) on DE-3 Sepharose 4^·
Kondicioniran medij, (glej primer 22), pripravimoConditioned medium (see example 22) is prepared
0,1 % z ozirom na Synperonic χη nato nanesemo na DE-30.1% with respect to Synperonic χη is applied to DE-3
Sepharoso ® . Po rahlem mešanju v teku 1 ure pri 4°C.Sepharose ® . After gentle mixing for 1 hour at 4°C.
izlijemo DE-3 Sepharoso 4B v kolono ter izpiramo z 0,2 (r)Pour DE-3 Sepharose 4B into the column and wash with 0.2 (r)
M NaCl, 0,1 % Synperonicc5 dokler UV absorpcija pri 280 nm ne doseže nivojev bazične linije, kar označuje odsotnostM NaCl, 0.1% Synperonicc 5 until UV absorption at 280 nm reaches baseline levels, indicating the absence
108 proteinov v eluatu. Z izpiranjem nato nadaljujemo z 0,2 M amonijevim acetatnim pufrom s pH 7,0, ki vsebuje 0,2 M amonijev tiocianat ter 0,1 % Synperonic . Potem ko UV adsorpcija pri 280 nm označuje odsotnost proteina v eluatu, eluiramo kolono z 0,2 M amonijevim acetatnim pufrom s pH 7,0, ki vsebuje 1,6 M amonijevega tiocianata in 0,1 % Synperonic®. Frakcije,/; ki vsebujejo najvišje amidolitične aktivnosti, izmerjene ob uporabi fluorometričnega poskusa s Cbz-Gly-Gly-Arg-AMC kot substratom /M. Zimmermann et al., Proč.Natl.Acad.Sci. USA. 75, 750 (1978)/, zberemo. Najmanj 80 % aktivnosti, vnesene v DE-3 Sepharose 4BVmaterial, pridobimo v eni sami konici (piku).108 proteins in the eluate. The washing is then continued with 0.2 M ammonium acetate buffer, pH 7.0, containing 0.2 M ammonium thiocyanate and 0.1% Synperonic®. After UV adsorption at 280 nm indicates the absence of protein in the eluate, the column is eluted with 0.2 M ammonium acetate buffer, pH 7.0, containing 1.6 M ammonium thiocyanate and 0.1% Synperonic®. The fractions/; containing the highest amidolytic activities, measured using a fluorometric assay with Cbz-Gly-Gly-Arg-AMC as substrate /M. Zimmermann et al., Proc.Natl.Acad.Sci. USA. 75, 750 (1978)/, are collected. At least 80% of the activity introduced into the DE-3 Sepharose 4BV material is recovered in a single peak (spot).
Zbrane aktivne frakcije dializiramo proti 0,2 M amonijevem acetatnem pufru s pH 7,0, ki vsebuje 0,1 % pThe collected active fractions were dialyzed against 0.2 M ammonium acetate buffer, pH 7.0, containing 0.1% p
Synperonic ter nanesemo na kolono, ki vsebuje monoklonalno protitelo- 4O7A.15.27, usmerimo proti prvi kringle” domeni ^r) t-PA, pripajamo na Sepharoso 48^, uravnotežimo v 0,2 M amonijevem acetatnem pufru s pH 7,0, ki vsebuje 0,1 %Synperonic and applied to a column containing monoclonal antibody-4O7A.15.27, directed against the first kringle domain ^r) t-PA, coupled to Sepharose 48^, equilibrated in 0.2 M ammonium acetate buffer pH 7.0 containing 0.1%
Synperonic®, da odstranimo endogeni t-PA. Zberemo efluent, ki vsebuje UK2TPAB(BC).Synperonic® to remove endogenous t-PA. Collect the effluent containing UK2 TPAB (BC).
Reverzna faza HPLC očiščenega UK2TPAB(BC) na koloni Nucleosir^ 3OO-5-C18 z dimenzijami 4 x 110 mm kaže eno samo konico pri eluciji z linearnim gradientom v teku 30 minut, izhajajoč iz 70 %-ne raztopine A, ki obstaja iz vode, katera vsebuje 0, 1 % trifluorocetne kisline, in 30 % raztopine B, ki obstaja iz acetonitrila, ki vsebuje 0,08 % trifluorocetne kisline, ter končamo s 40 % A in 60 % B.Reverse phase HPLC of purified UK2 TPAB (BC) on a Nucleosir^ 3OO-5-C18 column with dimensions 4 x 110 mm shows a single peak when eluting with a linear gradient over 30 minutes, starting from 70% solution A, consisting of water containing 0.1% trifluoroacetic acid, and 30% solution B, consisting of acetonitrile containing 0.08% trifluoroacetic acid, and ending with 40% A and 60% B.
109109
Očiščeni protein pokaže pri N-terminalni sekvenčni analizi prvih desetih aminokislinskih preostankov sekvenco SNELHQVPSN, ki je identična s sekvenco, ki bi jo pričakovali iz DNA sekvence, ki kodira molekulo.The purified protein shows, upon N-terminal sequence analysis of the first ten amino acid residues, the sequence SNELHQVPSN, which is identical to the sequence expected from the DNA sequence encoding the molecule.
PRIMER 24:EXAMPLE 24:
Čiščenje FK2UPAB(BC) in K2UPAB(BC)Cleaning FK2 UPAB (BC) and K2 UPAB (BC)
II
a. Priprava kolon z afiniteto do protitelesJ ffa. Preparation of antibody affinity columnsJ ff
Kunčja anti-uPA protitelesa* očiščena; iz kunčjega anti-uPA seruma, monoklonalna protitelesa^ 4O5B.33·3 in 4O6A.23.7, pripajamo naascc&anogenbromidom aktivirano Sepharose 4EM (Pharmacia), v skladu s proizvajalčevimi navodili, ob uporabi 6 mg npčotifeelesa/ml aktivirane Sepharose. Gelno matrico uravnotežimo s PBS, ki vsebuje 0,1 % Synperonicvo' in 0,1 % natrijevega azida.Rabbit anti-uPA antibodies* purified from rabbit anti-uPA serum, monoclonal antibodies^ 4O5B.33·3 and 4O6A.23.7, were coupled to ascorbate bromide-activated Sepharose 4EM (Pharmacia), according to the manufacturer's instructions, using 6 mg of nicotinamide/ml activated Sepharose. The gel matrix was equilibrated with PBS containing 0.1% Synperonicvo ' and 0.1% sodium azide.
b. Kromatografsko čiščenje FK2UPAB(BC) in K2UPAB(BC) na protitelesu/Sepharose 4Bb. Chromatographic purification of FK2 UPAB (BC) and K2 UPAB (BC) on antibody/Sepharose 4B
Kondicioniran medij (glej primer 22) naredimo (r)Conditioned medium (see example 22) is made (r)
0,1 % z ozirom na Synperonicv-> in ga nanesemo na anti-uPA Sepharose-4B ali na 4O5B.33.3 ali na 4O6A.23.7 Sepharose 4B. Zadnji dve protitelesi usmerimo proti drugi kringle domeni t-PA. Po rahlem mešanju v teku 2 ur pri 4°C izlijemo iprotitelo/Sepharoso v kolono in izperemo s PBS, ki vsebuje 1 M NaCl in 0,1 % Synperonic®, dokler UV absorpcija pri 280 nm ne označuje odsotnosti proteina v eluatu. Kolono nato eluiramo z 0,2 M glicin-HCl pufrom s pH 2,5. Frakcije0.1% with respect to Synperonicin -> and applied to anti-uPA Sepharose-4B or to 4O5B.33.3 or to 4O6A.23.7 Sepharose 4B. The latter two antibodies are directed against the second kringle domain of t-PA. After gentle mixing for 2 hours at 4°C, the antibody/Sepharose is poured onto the column and washed with PBS containing 1 M NaCl and 0.1% Synperonic® until the UV absorption at 280 nm indicates the absence of protein in the eluate. The column is then eluted with 0.2 M glycine-HCl buffer, pH 2.5. Fractions
110 zberemo v epruvetah, ki vsebujejo nevtralizirno količino110 are collected in test tubes containing a neutralizing amount
M Tris. Frakcije, ki vsebujejo najvišje amidolitične aktivnosti, izmerjene ob uporabi fluorometričnega poskusa s Cbz-Gly-Gly-Arg-AMC kot substratom /M. Zimmermann et al., Proč. Natl. Acad. Sci. USA. 75, 750 ( 1978)/, zberemo.M Tris. Fractions containing the highest amidolytic activities, measured using a fluorometric assay with Cbz-Gly-Gly-Arg-AMC as substrate (M. Zimmermann et al., Proc. Natl. Acad. Sci. USA. 75, 750 (1978)), were pooled.
nn
Rezervna faza HPLC- očiščenih FK2UPA (BC) in K2UPAB(BC) na Nueleosil® 3OO-5-C18 koloni z dimenzijami 4 x 110 mm, kaže vsakokrat po eno konico pri eluciji z linearnim gradientom v teku 30 minut, izhajajoč iz 70 %-ne raztopine A, ki obstaja iz vode, ki vsebuje 0,1 % trifluorocetne kisline, in 30 %-ne raztopine B, ki obstaja iz acetonitrila, ki vsebuje 0,08 % trifluorocetne kisline, ter končamo s 40 % A in 60 % B. N-končna sekvenčna analiza prvih petih preostankov očiščenih proteinov pokaže sekvenco SYQGN za K2UPAB(BC) in SYQVI za FK2UPAB(BC), ki je identična s sekvencami, ki bi jih pričakovali za DNA sekvence, ki kodirajo vsako molekulo.The reserve phase of HPLC-purified FK2 UPA (BC) and K2 UPAB (BC) on a Nueleosil® 3OO-5-C18 column with dimensions 4 x 110 mm each shows one peak when eluting with a linear gradient over 30 minutes, starting from 70% solution A, consisting of water containing 0.1% trifluoroacetic acid, and 30% solution B, consisting of acetonitrile containing 0.08% trifluoroacetic acid, and ending with 40% A and 60% B. N-terminal sequence analysis of the first five residues of the purified proteins shows the sequence SYQGN for K2UPAB (BC) and SYQVI for FK2UPAB (BC), which is identical to the sequences expected for the DNA sequences encoding each molecule.
PRIMER 25:EXAMPLE 25:
Poskus na aktivnost hibridnih plazminogenskih aktivatorjev v prisotnosti in odsotnosti fibrinogenskih fragmentovTesting the activity of hybrid plasminogen activators in the presence and absence of fibrinogen fragments
Uporabimo dvojni poskus, kot so ga opisali Verheyen et al. /Thromb. Haemost. 48., 266 ( 1982)/, ki sloni na konverziji plazminogena v plazmin s plazminogenskim aktivatorjem, čemur sledi reakcija plazmina s kromogenskim plazminskim substratom H-D-valil-L-levcil-L-lizin*p-*-nitroanilid dihidroklorid. Poskus izvedemo ria mikrotitrski plošči s 96 utori ter Titertek®* mikrotitrškim i ploščnim e i talni! kora. Utori vsebujejo 120-Χ ^ul 0,1 mol/1 Tris/HCl pufer s pH 7,5, ki vsebuje 0,1 % Tween 80, 20 ^ul Glu-piažminogen pri 1,3 /Umol/1, v zgoraj omenjenem Tris pufru, 100 /Ul plazminskega substrata z 0,7 mmoli/1 v Tris pufru, X yUl vzorec z znano koncentraci jo (X ustreza 10, 20, 40 oz. 60 ^ul), ali urokinazni standard z definirano aktivnostjo, izraženo v internacionalnih enotah (INT.U.) ter 10 ^ul stimulatorja (fibrinogenski fragmenti) s 3 mg/ml v destilirani vodi ali v 10 ^ul destilirane vode, če naj poskuse izvedemo brez stimulatorja. Povečana absorpcija svetlobe, deljena s kvadratom inkubacijskega časa, je sorazmerna aktivatorski aktivnosti plazminogena pri znani koncentraciji aktivatorja ter jo izrazimo v internacionalnih enotah. Kot standard uporabimo urokinazo -z vlsokčpmolekulsko maso in z definirano aktivnostjo, izraženo v internacionalnih enotah (t.j. INT.U. ; American Diagnostics). Vsakega izmed plazminogenskih aktivatorjev preizkusimo pri identičnih pogojih, v odsotnosti oz. prisotnosti fibrinogenskih fragmentov. Pri teh pogojih je dobljena razlika v aktivnostih merilo za stimulacijo plazminogenskih aktivatorjev s fibrinogenskimi fragmenti. Tabela 2 vsebuje rezultate analize, kar označuje odsotnost stimulacije za urokinazni standard v nasprotju s stimulacijo, ki jo izvajajo fibrinogenski fragmenti na molekule novega plazminogenskega aktivatorja, ki vsebujejo katalitsko domeno urokinaze. Ne glede na odsotnost ene ali več nekatalitskih domen F, G, K1 ali K2 tkivnega plazinogenskega aktivatorja, opazimo stimulacijo s fibrinogenskimi fragmenti za vse testirane hibridne molekule.We use a dual assay as described by Verheyen et al. /Thromb. Haemost. 48., 266 (1982)/, which is based on the conversion of plasminogen to plasmin by a plasminogen activator, followed by the reaction of plasmin with the chromogenic plasmin substrate H-D-valyl-L-leucyl-L-lysine*p-*-nitroanilide dihydrochloride. The assay is performed in a 96-well microtiter plate and Titertek®* microtiter plate and bottom! core. The wells contain 120-X µl of 0.1 mol/l Tris/HCl buffer, pH 7.5, containing 0.1% Tween 80, 20 µl Glu-plasminogen at 1.3 µmol/l in the above-mentioned Tris buffer, 100 µl of plasmin substrate at 0.7 mmol/l in Tris buffer, X µl of a sample with a known concentration (X corresponds to 10, 20, 40 or 60 µl), or a urokinase standard with a defined activity expressed in international units (INT.U.), and 10 µl of stimulator (fibrinogen fragments) at 3 mg/ml in distilled water or in 10 µl of distilled water if the experiments are to be performed without a stimulator. The increased light absorption divided by the square of the incubation time is proportional to the activator activity of plasminogen at a known concentration of activator and is expressed in international units. Urokinase is used as a standard - with a high molecular weight and a defined activity expressed in international units (i.e. INT.U.; American Diagnostics). Each of the plasminogen activators is tested under identical conditions, in the absence or presence of fibrinogen fragments. Under these conditions, the difference in activities obtained is a measure of the stimulation of the plasminogen activators by fibrinogen fragments. Table 2 contains the results of the analysis, which indicates the absence of stimulation for the urokinase standard in contrast to the stimulation exerted by fibrinogen fragments on the molecules of the novel plasminogen activator containing the catalytic domain of urokinase. Regardless of the absence of one or more of the non-catalytic domains F, G, K1 or K2 of tissue plasminogen activator, stimulation by fibrinogen fragments is observed for all hybrid molecules tested.
112 plasrainogen-aktivator112 plasminogen activator
---- <-· , --- ------- u-PA standard---- <-· , --- ------- u-PA standard
FGK2UPAB(BC) fk2upab(bc) k2upab fupabFGK2 UPAB (BC) fk2 upab (bc) k2 upab fupab
Tabela 2Table 2
I.U. stimuliran /I.U. nestimuliranI.U. stimulated /I.U. unstimulated
1,01.0
5,05.0
10,010.0
6,06.0
3,63.6
PRIMER 26:EXAMPLE 26:
Aktivnost mutantnih plazmlnogensklh aktivatorjev na llzo strdkovActivity of mutant plasminogen activators on clot formation
Aktivnosti na lizo strdkov ugotavljamo ob uporabi poskusa , kot sta ga opisala R.D. Philo in P.J. Gaffney /Thromb. Haemost. £5, 107-109 (1971)/. Logaritmični vnos liznega časa proti koncentraciji plazminogenskega aktivatorja je izražen v ravni črti. Specifično aktivnost plazminogenskega aktivatorja določimo s primerjavo s krivuljami, dobljenimi za standarden pripravek tkivnega plazminogenskega aktivatorja ali urokinaze.Clot lysis activities are determined using the assay described by R.D. Philo and P.J. Gaffney (Thromb. Haemost. £5, 107-109 (1971)). The logarithmic plot of lysis time versus plasminogen activator concentration is expressed as a straight line. The specific activity of plasminogen activator is determined by comparison with curves obtained for a standard preparation of tissue plasminogen activator or urokinase.
Krivulje vseh izmerjenih aktivatorjev imajo približno enak naklon, ki omogoči direktno zvezo med časom, potrebnim za lizo strdkov, in njihovo specifično aktivnostjo. Ker različni plazminogenski aktivatorji nimajo iste molekulske mase, morajo biti specifične aktivnosti izražene v molski koncentraciji namesto v običajni masni koncentraciji, da dobimo smiselne kriterije za učinkovitost različnih molekul. Ugotovimo, da je BThe curves of all measured activators have approximately the same slope, which allows a direct relationship between the time required for clot lysis and their specific activity. Since different plasminogen activators do not have the same molecular weight, specific activities must be expressed in molar concentration rather than in conventional mass concentration to provide meaningful criteria for the effectiveness of different molecules. We find that B
UK2TPA (BC) najmanj tako aktiven kot standarden t-PA,UK2 TPA (BC) at least as active as standard t-PA,
113 . plazmipogenski aktiva tor t-PA standard113. plasminogen activator t-PA standard
UK2TPA(BC) u-PA standard fgk2upab(bc) fkzupab(bc) k2upab(bc) fupab(bc) medtem ko FGK2UPAB(BC) in FK2UPAB(BC) kažeta aktivnosti, ki so skoraj enake t-PA, vendar znatno višje kot pri u-PA standardu.UK2 TPA(BC) u-PA standard fgk2 upab (bc) fkz upab (bc) k2 upab (bc) fupab (bc) while FGK2 UPAB (BC) and FK2 UPAB (BC) show activities that are almost the same as t-PA, but significantly higher than the u-PA standard.
gMr.
Ugotovimo, da ima KgUPA (BC) aktivnost, ki je skoraj identična u-PA standardu. Aktivnosti poskusa zberemo v tabeli 3·We find that KgUPA (BC) has an activity that is almost identical to the u-PA standard. The activities of the experiment are summarized in Table 3.
Tabela 3 j Endtesža:: lizo? s trdtoovJpmol·Table 3 j Endtesža:: lizo? s trdtoovJpmol·
23.6 28$623.6 28$ 6
13.213.2
24.324.3
IzFrom
20.720.7
10.410.4
2,7 * Enote za lizo strdkov uporabi molekulske mase izrazimo v pikomolih t-PA, ob t-PA na osnovi njene aminokislinske sekvence ter specifične aktivnosti 400.000 enot za lizo strdkov na mg.2.7 * Clot lysis units using molecular weight are expressed in picomoles of t-PA, with t-PA based on its amino acid sequence and specific activity of 400,000 clot lysis units per mg.
PRIMER 27:EXAMPLE 27:
Izpraznitev plazminogenskih aktivatorskih mutantnih molekul Iz obtoka kuncevClearance of Plasminogen Activator Mutant Molecules From the Circulation of Rabbits
1. Markiranje1. Marking
125125
Vse mutantne molekule radio-markiramo z J ob uporabi jodogenske metode /P.J. Fraker et al., Biochem.All mutant molecules were radiolabeled with I using the iodogenic method /P.J. Fraker et al., Biochem.
Biophys. Res. Commun. 80, 849-857 (1978)/.Biophys. Really. Commun. 80, 849-857 (1978)/.
125125
Da odstranimo prebitni prosti J mutantne molekule afinitetno očistimo bodisi ob uporabi metode, opisane v primeru 23 (PA imajo t-PA B-verigo) ali poTo remove excess free J mutant molecules are affinity purified either using the method described in Example 23 (PAs have a t-PA B-chain) or by
114 metodi, opisani v primeru 24 (PA imajo u-PA B-verigo.).114 methods described in Example 24 (PAs have a u-PA B-chain.).
Običajno dobimo specifične radioaktivnostiWe usually get specific radioactivities
2-20 /UCi//Ug proteina. Homogenost markiranih molekul določamo s SDS elektroforezo, ki ji sledi rentgenska avtoradiografija. V vseh primerih mutantne molekule migrirajo pri nereducirnih pogojih kot enojni trakovi in z Mr, ki so identični, kot pri nemarkiranih proteinih.2-20 /UCi//Ug of protein. The homogeneity of the labeled molecules is determined by SDS electrophoresis followed by X-ray autoradiography. In all cases, the mutant molecules migrate under non-reducing conditions as single bands and with Mr that are identical to those of the unlabeled proteins.
2. Študije izpraznitevi(Clearing)2. Clearing studies
Poskuse izvedemo na novozelandskih belih kuncih z maso 1,8 do 2,4 kg. Živali anestetiziramo subkutano s 1750 mg/kg Urethan^ (Merck, Darmstadt, ZRN). Izvedemo traheotomijo ter uvedemo cev iz umetne snovi v zunanjo vratno veno ter v skupno karotidno arterijo. 0,5 ml s fosfatom zapufrane slanice, ki vsebuje okoli 300 do 500 ng mutanta PA, injiciramo v vratno veno ter zapored odvzememamo serijske krvne vzorce (vsakokrat po 2 ml) ves čas v 60 minutnih intervalih preko karotidne arterije.Experiments were performed on New Zealand white rabbits weighing 1.8 to 2.4 kg. The animals were anesthetized subcutaneously with 1750 mg/kg Urethan^ (Merck, Darmstadt, Germany). A tracheotomy was performed and a plastic tube was inserted into the external jugular vein and the common carotid artery. 0.5 ml of phosphate-buffered saline containing approximately 300 to 500 ng of mutant PA was injected into the jugular vein and serial blood samples (2 ml each) were taken at 60-minute intervals via the carotid artery.
Krvne vzorce zberemo na citratu, takoj centrifugiramo s 3000 obrati na minuto v teku 15 minut in dekantiramo plazmo. AlikVote obarjamo v 10 %-ni triklorocetni kislini in preštejemo peletke v^-števcu.Blood samples were collected on citrate, immediately centrifuged at 3000 rpm for 15 minutes and the plasma decanted. Aliquots were precipitated in 10% trichloroacetic acid and the pellets were counted in a cytometer.
V primeri s t-PA, izoliranem iz celične linije Bowes melanoma, kažejo mutantne molekule naslednji razpolovni čas v obtoku.In the case of t-PA isolated from the Bowes melanoma cell line, mutant molecules exhibit the following half-lives in circulation.
hibridni PA razpolovni čas (min) za plasminogenski aktivator v obtokuhybrid PA half-life (min) for circulating plasminogen activator
115115
Vzorec izpraznitve t-PA je značilno bieksponencialen z zelo hitro Λ-fazo, čemur sledi počasnejša /^f-faza eliminacije. Eliminacija UK2TPAB(BC) in K2UPAB(BC) je skorajda monofazna, iz česar sklepamo, da je porazdelitev v drugi oddelek zavrta.The elimination pattern of t-PA is typically biexponential with a very rapid Λ-phase followed by a slower /^f-phase of elimination. The elimination of UK2 TPAB (BC) and K2 UPAB (BC) is almost monophasic, suggesting that distribution to the second compartment is inhibited.
3. Porazdelitev v organih3. Distribution in organs
Kunce obdelamo kot zgoraj. 20 minut po injekciji jodiranih mutantnih molekul kunce žrtvujemo? , j inf. odvzamemo glavne organe, določimo njihovo maso in po homogenizaciji alikvot preštejemo v/^-števniku.The rabbits are treated as above. 20 minutes after injection of the iodinated mutant molecules the rabbits are sacrificed? , j inf. the major organs are removed, their weight determined and after homogenization an aliquot is counted in a /^-counter.
Tabela 5 .Odstotek^ rekuperlrane radioaktivnosti t-PA jetra 40 srce < 1 pljuča <1 vranica < 1 ledvice uk2tpab(bc) < 1 < 1 < 1Table 5. Percentage^ of recovered radioactivity t-PA liver 40 heart < 1 lung < 1 spleen < 1 kidney uk2 tpab (bc) < 1 < 1 < 1
K2UPAB(BC) < 1 < 1 < 1K2 UPAB (BC) < 1 < 1 < 1
Mutantni PA kažejo večjo frakcijo radioaktivnih molekul, ki še vedno obstanejo v obtoku (supra), kar sovpada z močno zmanjšano izpraznitvijo iz jeter. Zmanjšan navzem v jetrih je zato pojasnilo za podaljšano razpolovnoMutant PAs show a higher fraction of radioactive molecules remaining in circulation (supra), which coincides with a greatly reduced clearance from the liver. Reduced hepatic uptake is therefore an explanation for the prolonged half-life.
116 dobo in monofazen eliminacijski vzorec mutantnih molekul, zlasti UK2TPAB(BC) in K2UPAB(BC).116 era and monophasic elimination pattern of mutant molecules, especially UK2 TPAB (BC) and K2 UPAB (BC).
nn
V primerih 28-34 uporabimo plazmide pCGC5/K2UPA pCGC6/FUPAB, pCGC7/FK2UPAB in pCGC8/FGK2UPAB (glej primer 18) za konstrukcijo kvasnih ekspresijskih plazmidov. Kvasna invertazna signalna sekvenca se v okviru zlije z različnimi kodirnimi sekvencami. Izražene so pod kontrolo inducirnega PH05 promotorja. Pri nekaterih konstruktih pa so mutirana glikozilaeijska mesta.In Examples 28-34, the plasmids pCGC5/K2 UPA pCGC6/FUPAB , pCGC7/FK2UPAB and pCGC8/FGK2UPAB (see Example 18) are used to construct yeast expression plasmids. The yeast invertase signal sequence is fused in frame to various coding sequences. They are expressed under the control of an inducible PH05 promoter. In some constructs, however, the glycosylation sites are mutated.
PRIMER 28:EXAMPLE 28:
Kloniranje izvora podvojitve faga F1 v ekspresijski vektor pJDB2O7Cloning of the F1 phage replication origin into the pJDB2O7 expression vector
Plazmidi-pEMBL družine /Dente et al., Nucl.Plasmids-pEMBL family /Dente et al., Nucl.
Acids Res. 11, 1645-55 (1983)/ vsebujejo področje genoma faga F1, ki nudi vse cis-učinku joče elemente za DNA podvojitev in morfogenezo. Samo po superinfekciji s fagom F1 (pomagaIni) se velike količine ienovlaknate plazmidne DNA izločijo v medij.Acids Res. 11, 1645-55 (1983)/ contain a region of the F1 phage genome that provides all the cis-acting elements for DNA replication and morphogenesis. Only after superinfection with F1 phage (helper) are large amounts of single-stranded plasmid DNA secreted into the medium.
Plazmid pEMBL19(+) se prebavi s Seal in EcoRI. Izolira se 2.2 kb fragment, ki vsebuje del na ampicilin rezistentnega gena pBR322 (mesto Seal), F1 intergensko območje in del d^-galaktozidaznega gena do polilinkerskega področja (mesto EcoRI).Plasmid pEMBL19(+) is digested with Sial and EcoRI. A 2.2 kb fragment is isolated containing part of the ampicillin resistance gene of pBR322 (Sial site), the F1 intergenic region and part of the d-galactosidase gene up to the polylinker region (EcoRI site).
Plazmid pJDB2O7 lineariziramo z razrezanjem s Hpal. 10 /Ug lineariziranega plazmida delno prebavimo sPlasmid pJDB2O7 was linearized by cutting with HpaI. 10 µg of the linearized plasmid was partially digested with
7,5 enotami EcoRI v prisotnosti 0,1 mg/ml homidijevega;: bromida v teku 40 minut pri 37°C. Reakcijo ustavimowith 7.5 units of EcoRI in the presence of 0.1 mg/ml of chromidium bromide for 40 minutes at 37°C. The reaction is stopped
117 z dodatkom 10 mM EDTA. Izoliramo 1.8 kb EcoRI-Hpal fragment na preparativnem 0,8 % agaroznem gelu.117 with the addition of 10 mM EDTA. The 1.8 kb EcoRI-HpaI fragment was isolated on a preparative 0.8% agarose gel.
yUg s Hpal razrezanega pJDB207 nadalje prebavimo 3 Seal. Izoliramo 4.8 kb veliki Hpal-Scal fragment. DNA fragmente elektroeluiramo z blokov agaroznega gela, očistimo z DE52 kromatografijo in obarjanjem z etanolom.yUg of HpaI-cut pJDB207 was further digested with 3 ScaI. A 4.8 kb HpaI-ScaI fragment was isolated. The DNA fragments were electroeluted from agarose gel blocks, purified by DE52 chromatography and ethanol precipitation.
0,2 pmola vsakega izmed 2.2 kb Scal-EcoRI fr*agjmenfeaf in 1.8 kb EcoRI-Hpal fragmenta in 0.1 pmol Hpal-Scal vektorskega fragmenta povežemo. Vezivno zmes uporabimo za transformacijo kompetentnih celic E.coli HB101 Ca2+.0.2 pmoles of each of the 2.2 kb Scal-EcoRI fragment and the 1.8 kb EcoRI-HpaI fragment and 0.1 pmole of the HpaI-Scal vector fragment were ligated. The ligation mixture was used to transform E.coli HB101 Ca2+ competent cells.
Plazmidno DNA 12, na' ampicilin rezistentnih, kolonij, analiziramo z dvojno prebavo z EcoRI/Pstl. Izberemo DNA enega samega klona s pravilnimi restrikcijskimi fragmenti in jo označimo kot pJDB207F1Lac.Plasmid DNA from 12 ampicillin-resistant colonies was analyzed by double digestion with EcoRI/PstI. DNA from a single clone with the correct restriction fragments was selected and designated pJDB207F1Lac.
PRIMER 29:EXAMPLE 29:
Konstrukcija plazmida pJDB207/PH05-I-FK2UPABBConstruction of plasmid pJDB207/PH05-I-FK2 UPAB B
Kodirno sekvenco FK2UPA ,prisotno v plazmidu B pCGC7/FK2UPA ,priredimo za izražanje v kvasu s fuzijo kvasne invertazne signalne sekvence in izraženjem gena pod kontrolo PH05 promotorja.The FK2 UPA coding sequence, present in plasmid B pCGC7/FK2 UPA, was adapted for expression in yeast by fusion to a yeast invertase signal sequence and expression of the gene under the control of the PH05 promoter.
Plazmid pCGC7/FK2UPA® (glej primer 18D) prebavimo s Pstl in BamHI. 1147 bp Pstl-BamHI fragment vsebuje BPlasmid pCGC7/FK2 UPA® (see Example 18D) was digested with Pstl and BamHI. The 1147 bp Pstl-BamHI fragment contains the B
FK2UPA , ki kodira sekvenco Pstl mesta na nukleotidnem položaju 199 t-PA (sl.1) na BamHI mesto v položaju 1322 u-PA (sl.3).FK2 UPA, which encodes the sequence PstI site at nucleotide position 199 of t-PA (Fig. 1) to BamHI site at position 1322 of u-PA (Fig. 3).
118 .118 .
Plazmid pJDB207/PH05-I-TPA (glej primer 6c) razrežemo s Šali in Pstl. 891 bp fragment izoliramo. Vsebuje PHO5 promotor, invertazno signalno sekvenco in 19 baz t-PA (Pstl mesto) .Plasmid pJDB207/PH05-I-TPA (see example 6c) was digested with SalI and PstI. An 891 bp fragment was isolated. It contained the PHO5 promoter, the invertase signal sequence and the 19 bases of t-PA (PstI site).
Plazmid pJDB207/PHO5-I-UPA (glej primer 8) prebavi vimo s Šali in BamHI. 6.6 kb vektorski fragment vsebuje 3’ del u-PA gena iz BamHI mesta v nukleotidnem položaju 1323 (sl. 3) proti položaju 1441 (PvuII mesto z dodanim Xhol li linkerjem) in PHO5 transkripcijske končne signale.Plasmid pJDB207/PHO5-I-UPA (see Example 8) was digested with SalI and BamHI. The 6.6 kb vector fragment contains the 3' part of the u-PA gene from the BamHI site at nucleotide position 1323 (Fig. 3) to position 1441 (PvuII site with added XhoII linker) and the PHO5 transcription termination signals.
0,2 pmola vsakega izmed 891 bp Sall-Pstl fragmenta in 1147 bp Pstl-BamHI fragmenta in 0,1 pmol 6,6 kb Sall-BamHI vektorskega fragmenta povežemo in uporabimo za transformacijo E.coli HB101 Ca2+ celic.0.2 pmoles of each of the 891 bp SalI-Pstl fragment and the 1147 bp Pstl-BamHI fragment and 0.1 pmoles of the 6.6 kb SalI-BamHI vector fragment were ligated and used to transform E. coli HB101 Ca2+ cells.
kolonij, rezistentnih na ampicilin, vzgojimo v LB mediju, ki vsebuje ampicilin (100 mg/1). Izoliramo-plazmid DNA in analiziramo z restrikcijsko prebavo z EcoRI in Hindlll. Izberemo en plazmid s pričakovanimi restrikcijskiD mi fragmenti in gajoznačimo kot pJDB207/PH05-I-FK2UPA .Ampicillin-resistant colonies were grown in LB medium containing ampicillin (100 mg/l). Plasmid DNA was isolated and analyzed by restriction digestion with EcoRI and HindIII. One plasmid with the expected restriction fragments was selected and designated pJDB207/PH05-I-FK2 UPA.
Plazmide pCGC6/EUPAB in pCGC8/FGK2UPAB se da uporabiti na enak način kot pCGC7/FK UPA . Nastali kvasni l ig ekspresijski plazmidi so označeni kot pJDB207 /PH05-I-FUPA oz. pJDB207/PHO5-I-FGKgUPAB.Plasmids pCGC6/EUPAB and pCGC8/FGK2 UPAB can be used in the same way as pCGC7/FK UPA . The resulting yeast l ig expression plasmids are designated pJDB207/PH05-I-FUPA and pJDB207/PHO5-I-FGKg UPAB , respectively.
PRIMER 30: Mutacija glikozilacijskega mesta pri /Asn 302/ urokinazne B-verigeEXAMPLE 30: Glycosylation site mutation at /Asn 302/ of urokinase B-chain
a) Kloniranje Pstl-BamHI fragmenta u-PA v m13mpl8:a) Cloning of the Pstl-BamHI fragment of u-PA into m13mpl8:
119119
Plazmid pJDB207 /PH05-I-UPA (glej primer 8), vsebuje popolno kodirno področje urokinaze. DNA razrežemo s Pstl in BamHI. 886 bp Pstl-BamHlffragment iz urokinaznega gena vsebuje gli kozilacijsko mesto (Asn 302) v nukleotidnih položajih 1033-1041. Nadaljnji fragment enake velikosti nadalje razraznežemo z BstEII. Izoliramo 886 bp Pstl.BamHI fragment na preparativnem 0,8%-nem agaroznem gelu.Plasmid pJDB207/PH05-I-UPA (see Example 8) contains the complete coding region of urokinase. The DNA was digested with Pstl and BamHI. The 886 bp Pstl-BamHI fragment from the urokinase gene contained a glycation site (Asn 302) at nucleotide positions 1033-1041. A further fragment of the same size was further digested with BstEII. The 886 bp Pstl.BamHI fragment was isolated on a preparative 0.8% agarose gel.
M13mpl8 RF-DNA razrežemo s Pstl in BamHI. 7.3 kb fragment izoliramo na preparativnem 0,8%-nem agaroznem gelu. DNA fragmente elektroeluiramo z agaroznega gela in očistimo z DE52 kromatografijo in obarjanjem z etanolom.M13mpl8 RF-DNA was digested with PstI and BamHI. The 7.3 kb fragment was isolated on a preparative 0.8% agarose gel. The DNA fragments were electroeluted from the agarose gel and purified by DE52 chromatography and ethanol precipitation.
0,1 pmol 7.3 kb PsfcI-BamHI razrezanega vektorja in 0.2 pmola 886 bp Pstl-BamHI u-PA fragmenta povežemo. En jil in 3 pl vezivne zmesi uporabimo za transformacijo celic E.coli JM108 Ca^+ v skladu s priročnikom M13 cloning and sequencing handbook obj. Amersham. Poberemo 12 brezbarvnih plikov ter pripravimo eno-vlaknato DNA /J. Messing.0.1 pmol of the 7.3 kb PsfcI-BamHI digested vector and 0.2 pmol of the 886 bp Pstl-BamHI u-PA fragment were ligated. One µl and 3 µl of the ligation mixture were used to transform E.coli JM108 Ca^+ cells according to the M13 cloning and sequencing handbook, vol. Amersham. 12 colorless plaques were collected and single-stranded DNA was prepared /J. Messing.
Methods in Enzymology 101, 21-78 (1983)/· Enovlaknato DNA uporabimo za pripravo delno dvcvlaknate DNA s sklenitvijo rMethods in Enzymology 101, 21-78 (1983)/· Single-stranded DNA is used to prepare partially double-stranded DNA by annealing.
ter podaljšanjem M13 univerzalnega primerja s Klenowo polimerazo. Reakcijski produkt ekstrahiramo s fenolom/kloroformom in DNA oborimo z etanolom. DNA razrežemo s Pstl in BamHI. 886 bp fragment označuje, da se je u-PA fragment kloniral v M13mpl8 vektorju. En klon nadalje analiziramo in pravilni insert potrdimo s sekvenciranjem. Klon označujemo kot M13mpl8/UPA.and extension of the M13 universal primer with Klenow polymerase. The reaction product was extracted with phenol/chloroform and the DNA was precipitated with ethanol. The DNA was digested with PstI and BamHI. The 886 bp fragment indicated that the u-PA fragment had been cloned into the M13mpl8 vector. One clone was further analyzed and the correct insert was confirmed by sequencing. The clone was designated M13mpl8/UPA.
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b) Mutacija glikozilacijskega mesta pri Asn302:b) Mutation of the glycosylation site at Asn302:
302 |Α3π!Ser Thr302 |Α3π!Ser Thr
M13mpl8 insert: 3’....AAA CCT TTT CTC TTA AGA TGG CTG ATA. ..5' (veriga v nasprotni smeri) mutagenski primer W: 5'-GGA AAA GAG CAA TCT ACC GAC-3’ veriga z muti- 5'....TTT GGA AAA GAG CAA TCT ACC GAC TAT...3’ rano smerjo: ,-, sefevencirni primer: CTGCCCTCGATGTATAACG 967 985M13mpl8 insert: 3’....AAA CCT TTT CTC TTA AGA TGG CTG ATA. ..5' (strand in the opposite direction) mutagenic primer W: 5'-GGA AAA GAG CAA TCT ACC GAC-3’ strand with muta- 5'....TTT GGA AAA GAG CAA TCT ACC GAC TAT...3’ direction: ,-, sevesion primer: CTGCCCTCGATGTATAACG 967 985
Sintetiziramo mutagenske in sekvencirne primerje ob uporabi fosforamiditne metode po /M.H. Caruthers, v: Chemical and Enzymatic Synthešis of Gene Fragmente, (ed. H.Mutagenic and sequencing primers are synthesized using the phosphoramidite method according to /M.H. Caruthers, in: Chemical and Enzymatic Synthesis of Gene Fragments, (ed. H.
G. Gassen and A. Lang) Verlag Chemie, Weinheim, ZRN/ na Applied Biosystem Model 38OB sintetizatorju.G. Gassen and A. Lang) Verlag Chemie, Weinheim, ZRN/ on an Applied Biosystem Model 38OB synthesizer.
In vitro mutagenezo na enovlaknati šabloni izvedemo tako, kot je opisal T.A. Kunkel /Proč. Nat. Acad. Sci. USA 82, 488-492 (1985)/. Proizvedemo enovlaknato šablono DNA, ki vsebuje uracil, z enim ciklom rasti na E.coli soju RZ1032 (dut~, ung”).In vitro single-stranded template mutagenesis was performed as described by T.A. Kunkel /Proc. Nat. Acad. Sci. USA 82, 488-492 (1985)/. A single-stranded DNA template containing uracil was produced by one cycle of growth on E. coli strain RZ1032 (dut~, ung”).
100 pmolov mutagenskega oligonukleotidnega primerja W fosforiliramo v 20 μΐ 50 mM Tris-HCl pH 7,5, 10 mM MgCl^ mM DTT, 0,5 mM ATP in 20 enotah T4 polinukleotidne kinaze (Boehringer) . Po 30 minutah pri 37°C ustavimo reakcijo s segrevanjem na 70°C v teku 10 minut.100 pmol of mutagenic oligonucleotide primer W is phosphorylated in 20 μΐ 50 mM Tris-HCl pH 7.5, 10 mM MgCl^ mM DTT, 0.5 mM ATP and 20 units of T4 polynucleotide kinase (Boehringer). After 30 minutes at 37°C, the reaction is stopped by heating to 70°C for 10 minutes.
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0,3 pmole M13mpl8/UPA šablone DNA, ki vsebuje uracil, inkubiramo z 10 pmoli fosforiliranega, mutagenskega, oligodezoksiribonukleotidnega primerja W in 10 pmoli Ml 3 univerzal nega sekvencirnega primerja ^030 μΐ 10 mM Tris-HCl pH 8,0 in 10 mM MgCl^. Vzorec segrejemo na 80°C in ga pustimo ohladiti do sobne temperature na majhni vodni kopeli.0.3 pmoles of M13mpl8/UPA template DNA containing uracil were incubated with 10 pmoles of phosphorylated, mutagenic, oligodeoxyribonucleotide primer W and 10 pmoles of M13 universal sequencing primer ^030 μΐ in 10 mM Tris-HCl pH 8.0 and 10 mM MgCl^. The sample was heated to 80°C and allowed to cool to room temperature in a small water bath.
c) Podaljševalno-vezivna reakcija:c) Extension-linkage reaction:
Zgornjemu r ena tur ir anemu vzorcu dodamo 10 μΐ zmesi encima-DNTP, ki vsebuje 1 mM dNTP, 10 mM Tris-HCl pH 8,0, 10 mM MgCl2, 20 mM DTT, 1 mM ATP, 400 enot T4 DNA ligaze (Bio=:labs, 400 U/μΙ) in 6 enot KlenovieDNA polimeraze (Boehrin^ ger, 6 U/jul). Inkubacija je pri 15°C čez noč.To the above purified sample, 10 μΐ of enzyme-dNTP mixture containing 1 mM dNTP, 10 mM Tris-HCl pH 8.0, 10 mM MgCl2 , 20 mM DTT, 1 mM ATP, 400 units of T4 DNA ligase (Bio=: labs, 400 U/μΙ) and 6 units of Klenow DNA polymerase (Boehringer, 6 U/μΙ) is added. Incubation is at 15°C overnight.
d) Transformacija celic E.coli BMH71:d) Transformation of E.coli BMH71 cells:
Vezivno zmes razredčimo na 200 μΐ s TE. Po 0,1 pl, 1 /iliin 10 /il podal j sevalno-vezivne zmesi dodamo h kompetentnim E.The binding mixture was diluted to 200 μΐ with TE. 0.1 μl, 1 μl, and 10 μl of the radiation-binding mixture were added to the competent E.
j coli BMH71 Ca celicam (Kunkel, supra). Po 30 minutah na ledu celice termošokiramo 3 minute pni 42°C in jih nato vzdržujemo na ledu. Celice prekrijemo z vrhnjim agarjem in E.coli JM101 indikatorskimi celicami.coli BMH71 Ca cells (Kunkel, supra). After 30 minutes on ice, the cells are heat shocked for 3 minutes at 42°C and then maintained on ice. The cells are overlaid with top agar and E. coli JM101 indicator cells.
plakov poberemo in jih uporabimo za inficiranje E.coli JM109. Fage izoliramo iz vrhnjega csloja (supernatanta) z obarjanjem s PEG. Enovlaknato DNA pripravimo z ekstrakcijo s fenolom in obarjanjem z etanolom. 'Šablone DNAcponovno suspendiramo v TE.Plaques were harvested and used to infect E.coli JM109. Phages were isolated from the supernatant by PEG precipitation. Single-stranded DNA was prepared by phenol extraction and ethanol precipitation. The template DNA was resuspended in TE.
Mutacijo AAT kodona (Asn302) do CAA kodona (Gln302) potrdimo za en klon z določitvijo DNA sekvence zThe mutation of the AAT codon (Asn302) to the CAA codon (Gln302) was confirmed for one clone by DNA sequencing with
122 zgoraj omenjenim sekvencirnim primerjem, ob uporabi metode za končanje verige /F. Sanger et al., Proč. Nat. Acad, Sci. USA 74, 5463-67 ( 1 977 /. Mutacija izzove Asn -> Gin spremembo v aminokislinskem položaju 302 u-PA in s tem eliminira eno samo glikozilacijsko mesto v urokinazi. W označuje mutacijo glikozilacijskega mesta v u-PA B-verigi (Asn302 ->Gln302 ). Pozitivni klon navajamo kot M13mp18/UPA-W.122 above-mentioned sequencing primers, using the chain termination method /F. Sanger et al., Proc. Nat. Acad, Sci. USA 74, 5463-67 ( 1 977 /. The mutation causes an Asn -> Gln change at amino acid position 302 of u-PA, thereby eliminating a single glycosylation site in urokinase. W indicates a mutation of the glycosylation site in the u-PA B-chain (Asn302 ->Gln302 ). The positive clone is referred to as M13mp18/UPA-W.
PRIMER 31: Transfer mutacije /Gln302/ v urokinazni βEXAMPLE 31: Transfer mutation /Gln302/ in urokinase β
B-verigi na FK^UPA hibrid:B-chains on FK^UPA hybrid:
Plazmid pJDB207/PH05-I-FK2UPAB prebavimo s Šali in Xhol. 2.2 kb Sall-Xhol fragment izolirajo, elektroeluiramo iz agaroznega gela, očistimo z DE52 kromatografijo in . ). r ko v a oborimo v etanolu. Ta DNA fragment vsebuje dve Mstl mesti v PH05 promotorju in u-PA sekvenco. 3 JJg 2.2 kb Sall-Xhol fragmenta delno prebavimo s 3 enotami Mstl v teku 10 minut pri 37°C. Reakcijskeppcodukte ločimo na preparativnem 0,8%nem agaroznem gelu ter izoliramo 1651 bp Sall-Mstl fragment ter ga elektroeluiramo z gela. DNA fragment vsebuje SallBamHI sekvenco pBR322, PH05 promotor, invertazno signalno βPlasmid pJDB207/PH05-I-FK2 UPAB was digested with SalI and XhoI. The 2.2 kb SalI-XhoI fragment was isolated, electroeluted from an agarose gel, purified by DE52 chromatography and ethanol precipitated. This DNA fragment contained two MstI sites in the PH05 promoter and a u-PA sequence. 3 µg of the 2.2 kb SalI-XhoI fragment was partially digested with 3 units of MstI for 10 minutes at 37°C. The reaction products were separated on a preparative 0.8% agarose gel and the 1651 bp SalI-Mstl fragment was isolated and electroeluted from the gel. The DNA fragment contained the SalI-BamHI sequence of pBR322, PH05 promoter, invertase signal β
sekvenco in FK2UPA kodirno sekvenco do Mstl mesta v u-PA delu v nukleotidnem položaju 935.sequence and the FK2 UPA coding sequence to the Mstl site in the u-PA portion at nucleotide position 935.
RF-DNA pripravimo za M13mp18/UPA-W (glej primer 30) s hitrim postopkom izolacije DNA /D.S. Holmes et al., Anai lyt. Biochem. 1 1 4, 1 93-97 ( 1 981 )/. 5 jig DNA prebavimo z BamHI in Mstl. Po dodatku 2 jig RNaze (Serva) in inkubiranjuRF-DNA was prepared for M13mp18/UPA-W (see Example 30) by the rapid DNA isolation procedure /D.S. Holmes et al., Analyt. Biochem. 1 1 4, 1 93-97 ( 1 981 )/. 5 µg of DNA was digested with BamHI and MstI. After addition of 2 µg of RNase (Serva) and incubation
123 minut pri 37°C 387 bp Mstl-BamHI fragment izoliramo na preparativnem 0,8%-nem agaroznem gelu. DNA fragment elektroeluiramo in oborimo v etanolu. Fragment vsebuje mutacijo AAT -> CAA pri nukleotidnih položajih 1033-1035 (Asn302-> Gin) v urokinazni B-verigi.123 minutes at 37°C. A 387 bp MstI-BamHI fragment is isolated on a preparative 0.8% agarose gel. The DNA fragment is electroeluted and ethanol precipitated. The fragment contains the AAT -> CAA mutation at nucleotide positions 1033-1035 (Asn302 -> Gln) in the urokinase B-chain.
Plazmid pJDB207/PH05-I-UPA razrežemo s Šali in BamHI. Izoliramo 6.6 kb vektorski fragment (glej primer 29). Povežemo 0,2 pmola 1651 bp Sall-Mstl fragmenta, 0,2 pmola 387 bp Mstl-BamHI fragmenta in 0,1 pmol 6.6 kb SallBamHI vektorskega fragmenta. Transformiramo kompetentne E.coli HB101 Ca^+ celice.Plasmid pJDB207/PH05-I-UPA was digested with SalI and BamHI. A 6.6 kb vector fragment was isolated (see Example 29). 0.2 pmole of the 1651 bp SalI-Mstl fragment, 0.2 pmole of the 387 bp Mstl-BamHI fragment and 0.1 pmole of the 6.6 kb SalI-BamHI vector fragment were ligated. Competent E.coli HB101 Ca^+ cells were transformed.
Vzgojimo 12 transformantov, rezistentnih na ampicilin. Plazmid DNA izoliramo in analiziramo z restrikcijskimi rezi z EcoRI in Hindlll. Mutacija (W) na glikozilacijskem mestu uniči EcoRI mesto v nukleotidnih položajih 1032—1037- Prisotnost mutacije je potrjena z DNA sekvenciranjem. Eno plazmidno DNA z mutacijo v u-PA B-verigi navajamo kot pJDB207/PHO5-I-FK2UPAB-W. Ta plazmid ima nedotaknjeno glikozilacijsko mesto v kringle K2, vendar mutantno mesto W(Asn302 ->· Gin) v u-PA B-verigi.Twelve ampicillin-resistant transformants were grown. Plasmid DNA was isolated and analyzed by restriction digestion with EcoRI and HindIII. The mutation (W) at the glycosylation site destroys the EcoRI site at nucleotide positions 1032—1037. The presence of the mutation was confirmed by DNA sequencing. One plasmid DNA with a mutation in the u-PA B-chain is designated pJDB207/PHO5-I-FK2 UPAB -W. This plasmid has an intact glycosylation site in kringle K2 , but a mutant site W(Asn302 ->· Gin) in the u-PA B-chain.
Plazmida pJDB207/PHO5-I-FUPAB-W in pJDB207/PHO5-I-FGK2UPAB-W konstruiramo na enak način, izhajajoč iz ustreznih, nemutiranih plazmidov (glej primer 29).Plasmids pJDB207/PHO5-I-FUPAB -W and pJDB207/PHO5-I-FGK2 UPAB -W were constructed in the same manner, starting from the corresponding, unmutated plasmids (see Example 29).
4.8. kb Sall-Hpal vektorski del pJDB207/PH05-IB4.8. kb SalI-HpaI vector portion of pJDB207/PH05-IB
FK2UPA —W nadomestimo s 6.2 kb Sall-Hpal vektorskim frag124 mentom pJDB207FlLac (glej primer 28). 6.2 kb fragment ima 1.4 kb FILac insert pEMBL19 kloniran na 4.8 kb fragment pJDB207. Po vezavi, transformaciji in analizi novega konstrukta navajamo en pravilen plazmid s FILac vključkom kot PJDB207F1Lac/PHO5-I-FK2UPAB-W.FK2 UPA —W is replaced with the 6.2 kb SalI-HpaI vector fragment pJDB207FlLac (see Example 28). The 6.2 kb fragment has the 1.4 kb FILac insert of pEMBL19 cloned into the 4.8 kb fragment of pJDB207. After ligation, transformation and analysis of the new construct, we refer to one correct plasmid with the FILac insert as PJDB207F1Lac/PHO5-I-FK2 UPAB -W.
Plazmid pJDB207F1Lac/PH05-I-FGK2UPAB-W dobimo na enak način.Plasmid pJDB207F1Lac/PH05-I-FGK2 UPAB -W was obtained in the same manner.
Na enak način 4.8 kb Sall-Hpal vektorski del pJDB207/PHO5-I-MOU-K2TPAB (glej primer 15C) nadomestimo s 6.2 kb Sall-Hpal vektorskim fragmentom pJDB207F1Lac. Nastali plazmid1 označimo kot pJDB207FlLac/PHQ5-I-UK2TPAB. Plazmide pJDB207F1Lac/PHQ5-I-UK2UPAB, pJDB207FlLac/PHQ5-ITPAAUPAB in pJDB207F1Lac/PHO5-I-UPAATPAB, dobimo na enak način iz plazmidov brez FILac vektorskega fragmenta..In the same way, the 4.8 kb Sal-HpaI vector fragment of pJDB207/PHO5-I-MOU-K2 TPAB (see Example 15C) was replaced with the 6.2 kb Sal-HpaI vector fragment of pJDB207F1Lac. The resulting plasmid1 was designated pJDB207FlLac/PHQ5-I-UK2TPAB . The plasmids pJDB207F1Lac/PHQ5-I-UK2UPAB , pJDB207FlLac/PHQ5-I-ITPAA UPAB and pJDB207F1Lac/PHO5-I-UPAA TPAB were obtained in the same way from plasmids without the FILac vector fragment.
PRIMER 32: Mutacija glikozilacijskega mesta /Asnl84GlySer/ v kringle K2 FK2UPAB-W:EXAMPLE 32: Mutation of the glycosylation site /Asnl84GlySer/ in kringle K2 FK2 UPAB -W:
a) Priprava enovlaknate šablone:a) Preparation of a single-fiber template:
Plazmid pJDB207F1Lac/PHO5-I-FK2UPAB-W uporabimo za transformacijo kompetentnih E.coli RZ1032 Ca^+ celic /T.A. Kunkel, supra/. Eno ampicilin rezistentno kolonijo gojimo v LB mediju, kateremu je dodano 100 jjg/ml ampicilina, 20 jjg/ml timidina in 20 /ig/ml dezoksiadenozina. Pri celični gPlasmid pJDB207F1Lac/PHO5-I-FK2 UPAB -W was used to transform competent E. coli RZ1032 Ca^+ cells /TA Kunkel, supra/. One ampicillin-resistant colony was grown in LB medium supplemented with 100 µg/ml ampicillin, 20 µg/ml thymidine and 20 µg/ml deoxyadenosine. At cell g
gostoti 1.10 /ml celice zberemo, izperemo v LB mediju in jih ponovno suspendiramo v LB mediju, ki vsebuje 100^Jg/ml ampicilina in 0,25 ^g/ml uridina. Pri Οϋ^θθ dodamo 0.3 pomagalnega faga R408 (Pharmacia-PL Biochemicals, Inc). vAt a density of 1.10 /ml, the cells are harvested, washed in LB medium and resuspended in LB medium containing 100 µg/ml ampicillin and 0.25 µg/ml uridine. At 0.3 µl, 0.3 µl of helper phage R408 (Pharmacia-PL Biochemicals, Inc) is added.
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m.o.i. 20. Kulturo krepko stresamo 5 ur pri 37°C. Uracil vsebujočo enovlaknato DNA v mediju izoliramo tako, kot je opisal T.A. Kunkel (zgoraj). Izhajajoč iz pEMBL19(+) (gl.m.o.i. 20. The culture was shaken vigorously for 5 hours at 37°C. Uracil-containing single-stranded DNA in the medium was isolated as described by T.A. Kunkel (above). Starting from pEMBL19(+) (see
primer 28) kloniramo F1 področje v pJDB207 v orientaciji, nasprotni -urnemu kazalcu. Izolirana enovlaknata DNA je B smiselno vlakno FK^UPA vključka v ekspresijskem plazmidu. b) Mutacija glikozilacijskega mesta pri Asnl84 kringle K2 t-PA:Example 28) Clone the F1 region into pJDB207 in a counterclockwise orientation. The isolated single-stranded DNA is the B sense strand of the FK^UPA insert in the expression plasmid. b) Mutation of the glycosylation site at Asn184 kringle K2 t-PA:
Mutacija se nanaša na tretji položaj konsenzne aminokislinske razpoznavne sekvence za glikozilacijo. Ser186 je nadomeščena z Ala.The mutation concerns the third position of the consensus amino acid recognition sequence for glycosylation. Ser186 is replaced by Ala.
.ŠS DNA:.SS DNA:
(veriga· s pravo usmeritvijo) mutagenskiprimer Y veriga z mutirano usmeritvijo sekvencirni primer(strand with correct orientation) mutagenic primer Y strand with mutated orientation sequencing primer
181» 186 Asn Gly|Ser]181» 186 Asn Gly|Ser]
Asn Gly[Ala|Asn Gly[Ala|
5'-CCACGGGAGGCAGGAGG-3 • ·5'-CCACGGGAGGCAGGAGG-3 • ·
791 775791 775
Mutacijski protokol je tak, kot je opisan v primeru 30. Namesto M13 univerzalnega sekvencirnega primerja uporabimo PHO5 oligonukleotidni primer s formuloThe mutation protocol is as described in Example 30. Instead of the M13 universal sequencing primer, we use the PHO5 oligonucleotide primer with the formula
5’-AGTCGAGGTTAGTATGGC-3’ ki hibridizira do nukleotidov -60 do -77 iz ATG v PHO5 promotorju. Po reakciji podaljšanja in vezave pretvorimo kompetentne E.coli BMH71 Ca^+ celice. /Kunkel, supra/. Ampicilin-rezistentne kolonije poberemo in gojimo v LB mediju, ki vsebuje 100 mg/1 ampicilina.5'-AGTCGAGGTTAGTATGGC-3' which hybridizes to nucleotides -60 to -77 from the ATG in the PHO5 promoter. After the extension and ligation reaction, competent E. coli BMH71 Ca^+ cells are transformed. /Kunkel, supra/. Ampicillin-resistant colonies are picked and grown in LB medium containing 100 mg/l ampicillin.
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Pripravimo plazmid DNA in analiziramo na prisotnost mutacije z DNA sekvenciranjem. Mutacija TCA kodona do GCA povzroči spremembo Ser -»- Ala v položaju amino kisline 186 t-PA.Plasmid DNA is prepared and analyzed for the presence of the mutation by DNA sequencing. Mutation of the TCA codon to GCA results in a Ser -»- Ala change at amino acid position 186 of t-PA.
Mutacija v tretjem položaju konsenzne sekvence odstrani glikozilacijsko mesto. En klon z mutirano DNA navajamo kot pJDB207F1Lac/PH05-I-FK2UPAB-WY.A mutation at the third position of the consensus sequence removes the glycosylation site. One clone with the mutated DNA is referred to as pJDB207F1Lac/PH05-I-FK2 UPAB -WY.
Y označuje mutacijo glikozilacijskega mesta pri Asnl84 v K2 t-PA in W mutacijo pri Asn302 v u-PA B-verigi.Y indicates a glycosylation site mutation at Asn184 in K2 t-PA and W a mutation at Asn302 in u-PA B-chain.
gMr.
Nastali neglikozilirani FK2UPA hibridni protein ima dve amino—kislinski spremembi: Serl86 -Ala v t-PA kringleThe resulting non-glycosylated FK2 UPA hybrid protein has two amino acid changes: Ser186 -Ala to t-PA kringle
K2 in Asn302 -r· Gin v u-PA B-verigi.K2 and Asn302 -r· Gin in the u-PA B-chain.
Analogna mutacija plazmida pJDB207F^Lac/PH05-IFGK2UPAB-W (glej primer 31) vodi do plazmida pJDB207RAn analogous mutation of the plasmid pJDB207F^Lac/PH05-IFGK2 UPAB -W (see example 31) leads to the plasmid pJDB207R
F1Lac/PH05-I-FK2UPA -WY, ki kodira za neglikozilirani FGK2UPAB hibridni protein.F1Lac/PH05-I-FK2 UPA -WY encoding a non-glycosylated FGK2 UPAB hybrid protein.
PRIMER 33: Konstrukcija plazmida pJDB207/PH05-I-K2UPAB-WY Nukleotidna sekvenca, ki kodira za hibridni K2~ gEXAMPLE 33: Construction of plasmid pJDB207/PH05-IK2 UPAB -WY Nucleotide sequence coding for hybrid K2 ~ g
UPA protein, kot je definiran z amino—kislinskim zaporedjem tPA(Ser1-Gln3)(Gly176-Arg275)-uPA(Ile159-Leu4l1) je vsebovana v plazmldu pCGC5/K2UPA . Za izražanje v kvasu uporabi mo inducirni PHO5 promotor ter zlijemo invertazno signalno gThe UPA protein, as defined by the amino acid sequence tPA(Ser1-Gln3)(Gly176-Arg275)-uPA(Ile159-Leu4l1) is contained in the plasmid pCGC5/K2 UPA . For expression in yeast, we use the inducible PHO5 promoter and fuse the invertase signal gene.
zaporedje v okvirju z K2UPA kodirnim področjem. Plazmid pCGC5/K2UPAB razrežemo z Bgll.I in AccI. Izoliramo 487 bp Bglll-AccI fragment. Vsebuje kodirno sekvenco iz Bglll mesta t-PA (nukleotidni položaj 178) na AccI mesto v u-PAsequence in frame with the K2 UPA coding region. Plasmid pCGC5/K2 UPAB was digested with Bglll.I and AccI. A 487 bp Bglll-AccI fragment was isolated. It contains the coding sequence from the Bglll site of t-PA (nucleotide position 178) to the AccI site of u-PA
127 (nukleotidni položaj 779). Fragment razrežemo s HphI, kar daje 4 fragmente.127 (nucleotide position 779). The fragment was cut with HphI, yielding 4 fragments.
Dva oligodezoksirobonukleotida s formulo Asn [Ala| (I) 5' -CTGCATCTTACCAAGGAAACAGTGACTGCTACTTTGGGAATGGGGCAGCCTACCGTGGCACG-3 ’ (II) 3'- AGAATGGTTCCTTTGTCACTGACGATGAAACCCTTACCCCGTCGGATGGCACCGTG -5' sintetiziramo ob uporabi fosforamiditne metode na sintetizatorju Applied Biosystem Model 38OB. Oligonukleotida I in II tvorita dvovlaknat DNA povezovalnik (linker). 5-nukleotidi pri odprtem 5’ koncu so del kvasne invertazne signalne sekvence, čemur sledi t-PA kodirna sekvenca (Serl-Gln3)(Glyl76-Thr191) do prvega HphI rezanega mesta v nukleotidnem položaju 752 (glej sl. 1). Glikozilacijsko mesto v položaju 729-737 (AsnGlySer) je mutirano v sintetični sekvenci od TCA (Ser) do GCA (Ala), s čemer odstranimo glikozilacijsko razpoznavno sekvenco. Mutacija glikozilacijskega mesta v aminokislinskih položajih 184-186 t-PA (npr. drugo glikozilacijsko mesto v izvirni t-PA) je označena kot Y.Two oligodeoxyribonucleotides with the formula Asn [Ala| (I) 5' -CTGCATCTTACCAAGGAAACAGTGACTGCTACTTTGGGAATGGGGCAGCCTACCGTGGGCACG-3' (II) 3'- AGAATGGTTCCTTTGTCACTGACGATGAAACCCTTACCCCGTCGGATGGCACCGTG -5' were synthesized using the phosphoramidite method on an Applied Biosystem Model 38OB synthesizer. Oligonucleotides I and II form a double-stranded DNA linker. The 5-nucleotides at the open 5' end are part of the yeast invertase signal sequence, followed by the t-PA coding sequence (Serl-Gln3)(Glyl76-Thr191) up to the first HphI cleavage site at nucleotide position 752 (see Fig. 1). The glycosylation site at position 729-737 (AsnGlySer) is mutated in the synthetic sequence from TCA (Ser) to GCA (Ala), thereby removing the glycosylation recognition sequence. The glycosylation site mutation at amino acid positions 184-186 of t-PA (i.e., the second glycosylation site in the native t-PA) is designated as Y.
128128
Oligonukleotide ' I in II fosforiliramo nannjihovih koncih,3segrevamo 10 minut pri 85°C in renaturiramo med ohlajevanjem na sobno temperaturo. 10,5 ^ug (270 pmolov) kinazirane, dvovlaknate linkerske DNA vežemo vOligonucleotides I and II are phosphorylated at their termini, heated for 10 minutes at 85°C and renatured while cooling to room temperature. 10.5 µg (270 pmol) of kinased, double-stranded linker DNA is ligated into
30-kratnem molskem prebitku na HphI rezane DNA fragmente (glej zgoraj), kot je opisano v primeru 8B. Prebitne linkerske molekule odstranimo z obarjanjem z izopropanolom. DNA nadalje prebavljamo s Seal. 252 bp fragment izoliramo na preparativnem 1,5 % agaroznem gelu, elektroeluiramo in obarjamo v etanolu.A 30-fold molar excess of HphI-cut DNA fragments (see above) was added as described in Example 8B. Excess linker molecules were removed by precipitation with isopropanol. The DNA was further digested with Sial. The 252 bp fragment was isolated on a preparative 1.5% agarose gel, electroeluted, and ethanol precipitated.
Plazmid P31RIT-12 (glej primer 6B) prebavimo s Sall in Xhol. Izolirani fragment nadalje prebavimo s Hgal (glej primer 60 in BamHI. Nastali 591 bp BamHI-Hgal fragment izoliramo. Vsebuje PH05 promotor in invertazno signalno sekvenco.Plasmid P31RIT-12 (see Example 6B) was digested with SalI and XhoI. The isolated fragment was further digested with Hgal (see Example 60) and BamHI. The resulting 591 bp BamHI-Hgal fragment was isolated. It contained the PH05 promoter and an invertase signal sequence.
Plazmid pJDB207/PH05-I-FK3UPAB-W prebavimo z BamHI. 5 ^ug linearne DNA delno prebavimo z 10 enotami Seal v teku 10 minut. Reakcijo ustavimo z dodatkom 10 mM EDTA. 7.7 kb BamHI-Scal vektorski fragment izoliramo, elektroeluiramo in oborimo v etanolu. Vsebuje 3’ delPlasmid pJDB207/PH05-I-FK3 UPAB -W was digested with BamHI. 5 µg of linear DNA was partially digested with 10 units of ScaI for 10 minutes. The reaction was stopped by adding 10 mM EDTA. The 7.7 kb BamHI-ScaI vector fragment was isolated, electroeluted and ethanol precipitated. It contains the 3' part
129 kodirne sekvence Seal mesta v t-PA (položaj 953) do konca u-PA B-verige (PvuII mesto v položaju 1441 z dodanim Xhol linkerjem), PH05 končne in pJDB2O7 vektorske sekvence.129 coding sequences Seal site in t-PA (position 953) to the end of u-PA B-chain (PvuII site at position 1441 with added XhoI linker), PH05 end and pJDB2O7 vector sequences.
0,2 pmola vsakega izmed 591 bp BamHI-Hgal fragmenta in 252 bp lepljivih koncev (linker)-Scal fragmenta in 0,1 pmol 7.7 kb vektorskega fragmenta povežemo. Po transformaciji E.coli HB101 Cad+ celic vzgojimo 12 ampicilin-rezistentnih kolonij. Plazmid DNA izoliramo in analiziramo z EcoRI in Hindlll prebavami. Prisotnost mutacij preverimo z DNA sekvenciranjem. Izberemo en pravilen klon in ga navajamo kot pJDB207/PH05-I-K2UPAB-WY. Glikozilirani mesti vakririgle K2 t-PA in v u-PA B-verigi sta obe mutirani (Y oz. W).0.2 pmoles of each of the 591 bp BamHI-Hgal fragment and the 252 bp sticky ends (linker)-Scal fragment and 0.1 pmole of the 7.7 kb vector fragment were ligated. After transformation of E.coli HB101 Cad+ cells, 12 ampicillin-resistant colonies were grown. Plasmid DNA was isolated and analyzed by EcoRI and HindIII digestions. The presence of mutations was checked by DNA sequencing. One correct clone was selected and designated as pJDB207/PH05-I-K2UPAB -WY. The glycosylated sites of the vacririgle K2 t-PA and in the u-PA B-chain were both mutated (Y and W, respectively).
gMr.
Nastali neglikozilirani I^UPA hibridni protein ima dve aminokislinski spremembi: Serl86 -> Ala v t-PAThe resulting non-glycosylated I^UPA hybrid protein has two amino acid changes: Ser186 -> Ala in t-PA
K2domeni in Asn302—>Gln v u-PA B-verigi.K2 domain and Asn302—>Gln in the u-PA B-chain.
PRIMER 34: Mutacija glikozilacijskih mest /Asnl84GlySer/ in /Asn448ArgThr/ v UK2TPAB hibriduEXAMPLE 34: Mutation of the glycosylation sites /Asnl84GlySer/ and /Asn448ArgThr/ in the UK2 TPAB hybrid
Uracil vsebujoča, enovlaknata šablona /T.A. Kunkel, supra/ plazmida pJDB207FlLac/PH05-IUK2TPAB (glej primer 31 )pripravimo tako, kot je opisano v primeru 30. Mutacijska shema za glikozilacijsko mesto pri Asnl84 je Xaka, kot je opisano v primeru 32. Mutacija glikozilacijskega mesta pri Asn448 povzroči aminokislinsko spremembo Thr450—>·The uracil-containing, single-stranded template (TA Kunkel, supra) of plasmid pJDB207FlLac/PH05-IUK2 TPAB (see Example 31) was prepared as described in Example 30. The mutation scheme for the glycosylation site at Asn184 is Xaka as described in Example 32. Mutation of the glycosylation site at Asn448 results in an amino acid change of Thr450—>·
Ala.Alas.
130 enovlaknata,DNA(vlaknojs Jravo usmeritvijo) mutagenski primer.Z:130 single-stranded,DNA (straight-stranded) mutagenic example. With:
vlaknoz mutirano usmeritvijo sekvencirni primer:fiber with mutated orientation sequencing example:
ι, Η 8 4 5 0ι, Η 8 4 5 0
Asn Arg|Thr|Asn Arg|Thr|
5'-...CTT AAC AGA ACA GTC ACC GAC A. ..-3'5'-...CTT AAC AGA ACA GTC ACC GAC A. ..-3'
3’-...GAA TTG TCT CGT CAG TGG CTG T...-5’3'-...GAA TTG TCT CGT CAG TGG CTG T...-5'
5*-...CTT AAC AGA GCA GTC ACC GAC A. ..-3'5*-...CTT AAC AGA GCA GTC ACC GAC A. ..-3'
Asn Arg[Ala|Asn Arg[Ala|
5'-TGGCAGGCGTCGTGCAA-3' • ·5'-TGGCAGGCGTCGTGCAA-3' • ·
1603 15871603 1587
Mutacijski protokol je opisan v primeru 30.The mutation protocol is described in Example 30.
Fosforilirana mutagenska primerja Y in Z sta oba renaturirana v uracil vsebujočo, enovlaknato šablono pJDB207FlLac/PH05 BPhosphorylated mutagenic primers Y and Z are both annealed to the uracil-containing, single-stranded template pJDB207FlLac/PH05 B
I-UK2TPA . V danem primeru uporabimo se PH05 oligonukleotidni primer (glej primer 32).I-UK2 TPA . In the given example, the PH05 oligonucleotide primer is used (see example 32).
Po podaljšanju in vezivni reakciji so transformirane kompetentne E.coli BMH71 Ca^4- celice. Pripravimo plazmid DNA ampilicin-rezistentnih transformantov in analiziramo na prisotnost obeh mutacij z DNA sekvenciranjem z označenimi sekvencirnimi primerji.After the extension and ligation reaction, competent E. coli BMH71 Ca^4- cells were transformed. Plasmid DNA of ampicillin-resistant transformants was prepared and analyzed for the presence of both mutations by DNA sequencing with labeled sequencing primers.
Plazmid DNA enega klona z obema mutacijama navajamo kot pJDB207FlLac/PH05-I-UK2TPAB-YZ. Y označuje mutacijo glikozilaeijskega mesta pri Asnl84 in Z mutacijo pri Asn448. Neglikozilirani UK2TPA hibridni protein ima dve aminokislinski spremembi: Serl86_> Ala v K2 kringle t-PA in Thr450—>Ala v t-PA B-verigi.The plasmid DNA of one clone with both mutations is designated as pJDB207FlLac/PH05-I-UK2 TPAB -YZ. Y denotes the glycosylation site mutation at Asn184 and Z the mutation at Asn448. The unglycosylated UK2 TPA hybrid protein has two amino acid changes: Ser186_> Ala in K2 kringle t-PA and Thr450—>Ala in t-PA B-chain.
Mutacijski protokol je uporaben tudi za šablone pJDB20 7F1 Lac/PH05-I-UKz UPA®, pJDB207FlLac/PH05-I-TPAAUPAB in pJDB2O7FlLac/PHO5-I-UPAATPAB (glej: primeri 31^' 7The mutation protocol is also applicable to the templates pJDB20 7F1 Lac/PH05-I-UKwith UPA®, pJDB207FlLac/PH05-I-TPAA UPAB and pJDB207FlLac/PHO5-I-UPAA TPAB (see: examples 31^' 7
131 z mutagenskim primerjem W za mutacijo glikozilacijakega mesta u-PA B-verige in /ali mutagenskega primer ja Y in Z in druge, objavljene v evropski patentni prijavi št. 225286, za mutacijo glikozilacijskih mest v t-PA.131 with mutagenic primer W for mutation of the glycosylation site of the u-PA B-chain and/or mutagenic primers Y and Z and others published in European Patent Application No. 225286 for mutation of glycosylation sites in t-PA.
PRIMER 35: Transformacija Saccharomyces cerevisiae GRF18 in priprava kvasnih celičnih ekstraktovEXAMPLE 35: Transformation of Saccharomyces cerevisiae GRF18 and preparation of yeast cell extracts
Plazmide pJDB2O7/PHO5-I-FK2UPAB.Plasmids pJDB2O7/PHO5-I-FK2 UPAB .
pJDB2O7FlLac/PHO5-I-FK2UPAB-W, pJDB207FlLac/PHO5-I-FK2UPAB-WY, pJDB207FlLac/PH05-I-UK2TPAB. pJDB2O7FlLac/PHO5-I-UK2TPAB-YZ, pJDB207/PHO5-I-K2UPAB-WY, pJDB207/PH05-I-FUPAB, pJDB207/PH05-I-FUPAB-W, pJDB2O7/PHO5-I-FGK2UPAB.pJDB207FlLac/PHO5-I-FK2 UPAB -W, pJDB207FlLac/PHO5-I-FK2UPAB -WY, pJDB207FlLac/PH05-I-UK2TPAB . pJDB2O7FlLac/PHO5-I-UK2 TPAB -YZ, pJDB207/PHO5-IK2 UPAB -WY, pJDB207/PH05-I-FUPAB , pJDB207/PH05-I-FUPAB -W, pJDB2O7/PHO5-I-FGK2 UPAB .
- pJDB207/PH05-I-FGK2UPAB-W.- pJDB207/PH05-I-FGK2 UPAB -W.
pJDB207FlLac/PHQ5-I-FGK2UPAB-W in pJDB2O7FlLac/PHO5-I-FGK2UPAB-WY transformiramo v Saccharomyces cerevisiae soj GRF18 (DSM 3665). Transformacija, celična rast in priprava celičnih ekstraktov so opisani v primeru 16.pJDB207FlLac/PHQ5-I-FGK2 UPAB -W and pJDB2O7FlLac/PHO5-I-FGK2 UPAB -WY were transformed into Saccharomyces cerevisiae strain GRF18 (DSM 3665). Transformation, cell growth and preparation of cell extracts are described in Example 16.
Nastale hibridne plazminogenske aktivatorje lahko očistimo na način, analogen onemu, opisanemu v primerih 22 do 24.The resulting hybrid plasminogen activators can be purified in a manner analogous to that described in Examples 22 to 24.
PRIMER 36: Priprava liofiliziranih, hibridnih plazminogenskih aktivatorjevEXAMPLE 36: Preparation of lyophilized, hybrid plasminogen activators
Raztopino, dobljeno po kateremkoli izmed primerov 22 do 24, nadalje očistimo in liofiliziramo kot sledi:The solution obtained according to any of Examples 22 to 24 is further purified and lyophilized as follows:
132 Raztopino razredčimo z 10 volumni 0,1 M amonijevega acetata s pH 5,0 (celokupni volumen 80 ml) in nanesemo na kolono, ki vsebuje 5 ml CM-Sepharoze Fast Flow (Pharmacia) s pretokom 25 ml/uro pri sobni temperaturi. Kolono smo predhodno uravnotežili z 0,1 M amonijevim acetatom. Perkolat, ki ne vsebuje produkta, zavržemo. Kolono izperemo s 15 ml 0,1 M amonijevega acetata s pH 5,0 in z 10 ml 0,1 M amonijevega acetata s pH 7,0. Nato izvedemo elucijo adsorbiranega hibridnega PA z 1 M amonijevim acetatom s pH 8,6 pri sobni temperaturi (pretok 5 ml/uro). Da preprečimo tvorbo plina na koloni izvedemo elucijo pri prebitnem tlaku 1 do 1,5 bar. Vsebnost hibridnega PA v eluatu merimo z UV monitorjem (280 nm). Zberemo frakcijo, ki vsebuje okoli 90 % eluiranega hibridnega:PA ter podvržemo liofilizaci ji. Čistoča trdnega hibridnega PA liofilizata je okoli ali nad 95 %, kot ocenimo s HPLC. Produkt ne vsebuje detergentov.132 The solution was diluted with 10 volumes of 0.1 M ammonium acetate pH 5.0 (total volume 80 ml) and applied to a column containing 5 ml of CM-Sepharose Fast Flow (Pharmacia) at a flow rate of 25 ml/hour at room temperature. The column was previously equilibrated with 0.1 M ammonium acetate. The percolate containing no product was discarded. The column was washed with 15 ml of 0.1 M ammonium acetate pH 5.0 and 10 ml of 0.1 M ammonium acetate pH 7.0. The adsorbed hybrid PA was then eluted with 1 M ammonium acetate pH 8.6 at room temperature (flow rate 5 ml/hour). To prevent gas formation on the column, the elution was carried out at an excess pressure of 1 to 1.5 bar. The content of hybrid PA in the eluate is measured with a UV monitor (280 nm). A fraction containing about 90% of the eluted hybridPA is collected and subjected to lyophilization. The purity of the solid hybrid PA lyophilizate is about or above 95%, as assessed by HPLC. The product does not contain detergents.
PRIMER 37: Prvi farmacevtski sestavek za parenteralno dajanjeEXAMPLE 37: First Pharmaceutical Composition for Parenteral Administration
Raztopino, ki vsebuje čisti uPA(1-44)-tPA(176-527), dobljen kot je opisano zgoraj, dializiramo proti 0,3 molarnem natrijevem kloridu, ki vsebuje 0,01 % Tween 80™ in shranimo pri -80°C. Pred dajanjem nastavimo koncentracijo na 75/Ug/ml celokupne PA in 0,3 M NaCl. Raztopino steriliziramo s filtracijo skozi 0,22 ^um membranski filter.The solution containing pure uPA(1-44)-tPA(176-527), obtained as described above, was dialyzed against 0.3 molar sodium chloride containing 0.01% Tween 80™ and stored at -80°C. Prior to administration, the concentration was adjusted to 75 µg/ml total PA and 0.3 M NaCl. The solution was sterilized by filtration through a 0.22 µm membrane filter.
Namesto zgoraj omenjenega PA lahko uporabimo tudi isto količino drugih PA, opisanih v prejšnjih primerih, kot npr.Instead of the above-mentioned PA, we can also use the same amount of other PAs described in the previous examples, such as
-133 uPA(1-158)-tPA(276-527), uPA( 1-131)-tPA(263-527), tPA(l-275)-uPA(159-411) , tPA(l-262)-uPA(132-411), uPA(l-44)-tPA(176-261)-uPA(134-411), tPA( 1-49)-tPA(262-275)-uPA(159-411), tPA(1-49)-uPA(134-411), tPA(l-49)-tPA(176-275)-uPA(159-411), tPA(l-49)-tPA(176-262)-uPA(132-411), tPA(l-3)-tPA(176-275)-uPA(159-411), tPA(l-86)-tPA(176-275)-uPA(159-411) ali tPA(l-86)-tPA(176-262)-uPA(132-411), ali mutanjtni hibrijdnSiPA', kot npr. - _ tPA(l-49)-tPA(262-275)-uPA(159-301, Glnf 303-411), tPA(l-49)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411), uPA(l-44)-tPA(176-185, Ala, 187-449, Ala, 451-527), tPA(l-3)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411) ali tPA(l-86)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411). ,-133 uPA(1-158)-tPA(276-527), uPA(1-131)-tPA(263-527), tPA(l-275)-uPA(159-411), tPA(l-262)-uPA(132-411), uPA(l-44)-tPA(176-261)-uPA(134-411), tPA(1-49)-tPA(262-275)-uPA(159-411), tPA(1-49)-uPA(134-411), tPA(l-49)-tPA(176-275)-uPA(159-411), tPA(1-49)-tPA(176-262)-uPA(132-411), tPA(l-3)-tPA(176-275)-uPA(159-411), tPA(l-86)-tPA(176-275)-uPA(159-411) or tPA(l-86)-tPA(176-262)-uPA(132-411), or mutant hybrids such as SiPA'. - _ tPA(l-49)-tPA(262-275)-uPA(159-301, Glnf 303-411), tPA(l-49)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411), uPA(l-44)-tPA(176-185, Ala, 187-449, Ala, 451-527), tPA(l-3)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411) or tPA(l-86)-tPA(176-185, Ala, 187-275) 187-275)-uPA(159-301, Gin, 303-411). ,
PRIMER 38: Drugi farmacevtski sestavek za parenteralno dajanje (disperzija za injiciranje)EXAMPLE 38: Second Pharmaceutical Composition for Parenteral Administration (Injectable Dispersion)
169,3 mg sojinega lecitina (sojin fosfatit NC169.3 mg soy lecithin (soy phosphatite NC
95, proizvajalec: Nattermann, Koeln, ZRN, čistoča 90 do %; sestava maščobnih kislin: linolne kisline 61 do 71 %, linolenske kisline 4 do 7 %, oljeve kisline 6 do 13 %, palmitinske kisline 10 do 15 %, stearinske kisline 1,5 do95, manufacturer: Nattermann, Koeln, ZRN, purity 90 to %; fatty acid composition: linoleic acid 61 to 71%, linolenic acid 4 to 7%, oleic acid 6 to 13%, palmitic acid 10 to 15%, stearic acid 1.5 to
3,5 %) in 92,7 mg čistega natrijevega glikoholata raztopimo v 752,5 ml sterilizirane vode. Raztopino nastavimo na pH 7,4 z 1 N NaOH. Dodamo 10 mg .liofiliziranega uPA(1-44)-tPA(176527). Zmes mešamo, dokler ne dobimo bistre raztopine.3.5%) and 92.7 mg of pure sodium glycocholate are dissolved in 752.5 ml of sterilized water. The solution is adjusted to pH 7.4 with 1 N NaOH. 10 mg of lyophilized uPA(1-44)-tPA(176527) are added. The mixture is stirred until a clear solution is obtained.
Raztopino steriliziramo s filtracijo skozi 0,22 ^um membranski filter in napolnimo v ampule.The solution is sterilized by filtration through a 0.22 µm membrane filter and filled into ampoules.
Namesto zgoraj omenjenega PA lahko uporabimo tudi enako količino :nekega drugega pa, opisanega v prejšnjih primerih, kot npr.Instead of the above-mentioned PA, we can also use the same amount of another PA described in the previous examples, such as e.g.
- 134 uPA(1-158)-tPA(276-527), uPA(l-131)-tPA(263-52'7) , tPA(l-275)-uPA(159-411), tPA(1-262)-uPA(132-411), uPA(l-44)-tPA(176-261)-uPA(134-411) , tPA(l-49)-tPA(262-275)-uPA(159-411) , tPA(l-49)-uPA( 134-411) , tPA(l-49)-tPA(176-275)-uPA(159-4ll), tPA(l-49)-tPA(176-262)-uPA(132-411), tPA(l-3)-tPA(176-275)-uPA(159-411), tPA(1-86)-tPA(176-275)-uPA(159-411) ali tPA(l-86)-tPA(176-262)-uPA(132-411), ali mutar^triiruhibr^dni PA,’;-kot '«npcfi- ’ .-ko- ' nj d tPA(l-49)-tPA(262-275)-uPA(159-301, Gin, 303-411), tPA(l-49)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411), uPA(l-44)-tPA(176-185, Ala, 187-449, Ala, 451-527), tPA(l-3)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411) ali tPA(l-86)-tPA(176-185, Ala, 187-275)-uPA(159-3Ol, Gin, 303-411).- 134 uPA(1-158)-tPA(276-527), uPA(1-131)-tPA(263-52'7), tPA(1-275)-uPA(159-411), tPA(1-262)-uPA(132-411), uPA(l-44)-tPA(176-261)-uPA(134-411), tPA(l-49)-tPA(262-275)-uPA(159-411), tPA(l-49)-uPA(134-411), tPA(1-49)-tPA(176-275)-uPA(159-4ll), tPA(l-49)-tPA(176-262)-uPA(132-411), tPA(l-3)-tPA(176-275)-uPA(159-411), tPA(1-86)-tPA(176-275)-uPA(159-411) or tPA(l-86)-tPA(176-262)-uPA(132-411), or mutar^triiruhibr^dni PA,';-as '«npcf i- ' .-ko- ' nj d tPA(l-49)-tPA(262-275)-uPA(159-301, 303-411), Gin, tPA(1-49)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411), uPA(l-44)-tPA(176-185, Ala, 187-449, Ala, 451-527), tPA(l-3)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gin, 303-411) or tPA(1-86)-tPA(176-185, Ala, 187-275)-uPA(159-301, Gn, 303-411).
PRIMER 39: Tretji farmacevtski sestavek za parenteralno dajanje (vključno bolusno injiciranje)EXAMPLE 39: Third Pharmaceutical Composition for Parenteral Administration (Including Bolus Injection)
100 mg hibridnega plazminogenskega aktivatorja ali mutantnega hibridnega plazminogenskega aktivatorja, npr. takega, kot so omenjeni v primerih 37 in 38, raztopimo v 1000 ml 50 mM glutaminske kisline/natrijev glutamat, ki vsebuje 0,7 % NaCl, s pH 4,5. Raztopino napolnimo v ampule in jo lahko uporabimo za intravenozno (bolusno ) infuzijo.100 mg of hybrid plasminogen activator or mutant hybrid plasminogen activator, e.g. as mentioned in Examples 37 and 38, is dissolved in 1000 ml of 50 mM glutamic acid/sodium glutamate containing 0.7% NaCl, pH 4.5. The solution is filled into ampoules and can be used for intravenous (bolus) infusion.
Deponiranje mikroorganizmovDeposition of microorganisms
Naslednje soje smo deponirali leta 1987 pri Deutsche Sammlung fiir Mikroorganismen (DSM), Grisebachstrasse 8, D-3000 Gottingen ZRN (z danimi pristopnimi števili):The following strains were deposited in 1987 at the Deutsche Sammlung fiir Mikroorganismen (DSM), Grisebachstrasse 8, D-3000 Gottingen ZRN (with accession numbers given):
135 Pristopno št.135 Accession no.
E.coli HB101/pW349FE. coli HB101/pW349F
E.coli HB101/pCSl6E. coli HB101/pCSl6
E.coli HB101/pcUKl76E.coli HB101/pcUKl76
E.coli HBl01/pCGA26E. coli HBl01/pCGA26
E.coli HB101/pSV2911neo.E. coli HB101/pSV2911neo.
Naslednje hibridomske celične linije smo deponirali leta 1987 pri Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Pariš (CNCM) Francija, pod danimi pristopnimi števili.The following hybridoma cell lines were deposited in 1987 at the Collection Nationale de Cultures de Microorganismes, Institut Pasteur, Paris (CNCM) France, under the accession numbers given.
hibridom! Pristopno št.hybrids! Accession no.
405B.33.3405B.33.3
406A.23.7406A.23.7
407A.15.27407A.15.27
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| YU00195/88AYU19588A (en) | 1988-02-01 | 1988-02-01 | Hybride proteins |
| Publication Number | Publication Date |
|---|---|
| SI8810195Atrue SI8810195A (en) | 1996-08-31 |
| SI8810195B SI8810195B (en) | 1998-10-31 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SI8810195ASI8810195B (en) | 1988-02-01 | 1988-02-01 | Hybride proteins |
| Country | Link |
|---|---|
| SI (1) | SI8810195B (en) |
| YU (1) | YU19588A (en) |
| Publication number | Publication date |
|---|---|
| YU19588A (en) | 1991-06-30 |
| SI8810195B (en) | 1998-10-31 |
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| Date | Code | Title | Description |
|---|---|---|---|
| IF | Valid on the event date |