DK170346B1 - Granulocyte Macrophage Colony Stimulating Factor (CSF) Proteins, Method for Preparation thereof, Expression Vector and E.coli Cell for Use in This Method and Using These Proteins - Google Patents
Granulocyte Macrophage Colony Stimulating Factor (CSF) Proteins, Method for Preparation thereof, Expression Vector and E.coli Cell for Use in This Method and Using These Proteins Download PDFInfo
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Abstract
Description
i DK 170346 B1in DK 170346 B1
Human granulocyt-makrofag-"Colony-Stimulating,,-f aktor (GM-CSF), i det følgende kaldet "CSF", er et glycoprotein med en molvægt på ca. 23.000 Dalton. cDNA-sekvensen og ekspressionen af glycoproteinet i pattedyreceller er allerede 5 kendt (G.G. Wong et al., Science 228 (1985), 810-815, D.Human granulocyte macrophage "Colony-Stimulating," - factor (GM-CSF), hereinafter referred to as "CSF", is a glycoprotein having a molecular weight of about 23,000 Daltons. The cDNA sequence and expression of the glycoprotein in mammalian cells is already known (GG Wong et al., Science 228 (1985), 810-815, D.
Métcalf, Science 229 (1985), 16-22).Metcalf, Science 229 (1985), 16-22).
Opfindelsen angår gramilocyt-makrofag-"Colony Stimu-lating"-faktor(CSF)-proteiner, som er tilgængelige ved i og for sig kendte variationer af DNA-sekvenserne. Således kan 10 man eksempelvis ved konstruktionen af vektorer til fusionsproteiner indbygge spaltesteder, som efter fraspaltning af CSF-proteinet viser C-terminale og/eller N-terminale variationer i aminosyresekvensen. Opfindelsen angår også en fremgangsmåde til fremstilling af CSF-proteiner ifølge opfindel-15 sen. Desuden angår opfindelsen anvendelsen af CSF-proteiner ifølge opfindelsen til fremstilling af lægemidler samt lægemidler, som indeholder eller består af CSF-proteiner ifølge opfindelsen, især lægemidler til stimulering af proliferatio-nen af haematopoietiske celler og til fremme af ganulocyt-20 og makrofag-dannelsen.The invention relates to gramilocyte macrophage "Colony Stimulation" factor (CSF) proteins, which are available by known variations of the DNA sequences. Thus, for example, in the construction of vectors for fusion proteins, cleavage sites can be incorporated which, after cleavage of the CSF protein, show C-terminal and / or N-terminal variations in the amino acid sequence. The invention also relates to a method for producing CSF proteins according to the invention. In addition, the invention relates to the use of CSF proteins of the invention for the manufacture of drugs and drugs containing or consisting of CSF proteins of the invention, in particular drugs for stimulating the proliferation of haematopoietic cells and for promoting ganulocyte-20 and macrophage formation. .
Opfindelsen angår desuden ekspressionsvektorer til anvendelse i bakterier, især i E. coli, som indeholder en for CSF eller et CSF-fusionsprotein kodende DNA i egnet orden ("operatively linked to") .The invention further relates to expression vectors for use in bacteria, especially in E. coli, which contain a DNA operatively linked to CSF or CSF fusion protein.
25 Yderligere aspekter af opfindelsen og foretrukne udførelsesformer derfor belyses nærmere i det følgende eller defineres i patentkravene.Further aspects of the invention and preferred embodiments are therefore elucidated in the following or defined in the claims.
Til belysning af opfindelsen tjener endvidere figurerne 1-15 på tegningen, hvor man i hvert enkelt tilfælde, for 30 det meste i form af et rutediagram, anskueliggør fremgangsmåderne i eksemplerne med det samme nummer. Disse figurer er ikke i korrekt målestok, især i polylinkerområdet er målestokken "forlænget".In order to illustrate the invention, figures 1-15 of the drawing are also used, in which in each case, mostly in the form of a flow chart, the methods of the examples with the same number are illustrated. These figures are not on the correct scale, especially in the polylinker range the scale is "extended".
Således vises der i fig. 1 og fortsættelserne 35 la og lb deraf fremstillingen af vektoren pW 225, som tjener til direkte ekspression af (Met-)CSF. De følgende 2 DK 170346 B1Thus, in FIG. 1 and sequences 35 1a and 1b thereof, the preparation of the vector pW 225, which serves to directly express (Met-) CSF. The following 2 DK 170346 B1
OISLAND
figurer angår vektorer, som leder til ekspression af fusionsproteiner, i hvis N-terminal der er anbragt et "ballast"--protein foran CSF-aminosyresekvensen, og dette protein er afledt af en delsekvens af det humane interleukin-2, 5 i det følgende betegnet "IL-2" eller "/^EL-2": * I fig. 2 og fortsættelserne 2a og 2b deraf vises fremstillingen af vektoren pW 216, der koder for et fusionsprotein, ud fra hvilket der ved syrespaltning fremstår et CSF-derivat, som N-terminal er forlænget med 10 aminosyren prolin.Figures relate to vectors leading to expression of fusion proteins in whose N-terminal a "ballast" protein is placed in front of the CSF amino acid sequence, and this protein is derived from a human sequence interleukin-2,5 subunit below. designated "IL-2" or "/ ^ EL-2": * In FIG. 2 and sequences 2a and 2b thereof, the preparation of the vector pW 216 encoding a fusion protein is shown from which, upon acid cleavage, a CSF derivative, which is N-terminal, is extended by the 10 amino acid proline.
I fig. 3 vises syntesen af vektoren pW 240, der koder for et fusionsprotein, som efter syrespaltning leder til et CSF-derivat, som i stedet for den faste aminosyre (alanin) bærer prolin.In FIG. 3 shows the synthesis of the vector pW 240, which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative which carries the proline instead of the solid amino acid (alanine).
15 Fig. 4 angår fremstillingen af vektoren pW 241, som koder for et fusionsprotein, der efter syrespaltning leder til et CSF-derivat, hos hvilket den første aminosyre (alanin) mangler.FIG. 4 relates to the preparation of the vector pW 241, which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative in which the first amino acid (alanine) is missing.
I fig. 5 vises fremstillingen af vektoren pW 242, 20 som koder for et fusionsprotein, som efter syrespaltning leder til et CSF-derivat, i hvilket de første 5 aminosyrer er fjernede.In FIG. 5 shows the preparation of the vector pW 242, 20 which encodes a fusion protein which, after acid cleavage, leads to a CSF derivative in which the first 5 amino acids are removed.
Fig. 6 angår fremstillingen af vektoren pW 243, der koder for et fusionsprotein, hvilket efter syrespaltning 25 leder til et CSF-derivat, i hvilket de første 7 aminosyrer mangler.FIG. 6 relates to the preparation of the vector pW 243 encoding a fusion protein which, after acid cleavage, 25 leads to a CSF derivative in which the first 7 amino acids are missing.
I fig. 7 vises syntesen af vektoren pW 244, der koder for et fusionsprotein, ved hjælp af hvilket man efter syrespaltning får et CSF-derivat, i hvilket 30 de første 11 aminosyrer er eliminerede.In FIG. 7, the synthesis of the vector pW 244 encoding a fusion protein is shown by which, after acid cleavage, a CSF derivative is obtained in which the first 11 amino acids are eliminated.
I fig. 8 og fortsættelsen 8a deraf vises synteten af vektoren pW 246. Denne koder for et fusionsprotein, i hvilket der efter IL-2-delsekvensen følger to varierede, som "CSF"' betegnede sekvenser. Efter 35 syrespaltning får man et CSF-derivat, i hvilket prolin er N-terminaltforan den første aminosyre, og i hvilket den 3 DK 170346 B1 0..In FIG. 8 and continuation 8a thereof, the synthesis of the vector pW 246 is shown. This encodes a fusion protein in which, following the IL-2 sub sequence, two variants are designated as "CSF" designated sequences. After acid cleavage, a CSF derivative is obtained in which proline is the N-terminal front of the first amino acid and in which it is.
sidste aminosyre er udskiftet med asparaginsyre.last amino acid has been replaced with aspartic acid.
I fig. 9 vises syntesen af vektoren pW 247/ som koder for et fusionsprotein, i hvilket der efter IL-2-delsekyensen følger tre CSP'-sekvenser. Efter syre-5 spaltning fås det i fig. 8 karakteriserede CSF-derivat.In FIG. 9, the synthesis of the vector pW 247 / which encodes a fusion protein is shown in which, following the IL-2 sub-sequence, three CSP 'sequences follow. After acid cleavage, it is obtained in FIG. 8 characterized CSF derivative.
I fig. 10 og fortsættelsen, fig. 10a, deraf vises fremstillingen af hybridplasmiderne pS 200-204, som har syntetiske CSF-DNA-delsekvenser, idet plasmidet pS 200 indeholder "Synteseblok I" ifølge bilag I, plasmidet pS 201 10 indeholder "Synteseblok II" ifølge bilag II, plasmidet pS 202 indeholder "Synteseblok III"’’ ifølge bilag III, plasmidet pS 203 indeholder hele det syntetiske gen, og pS 204 er et ekspressionsplasmid, som ligeledes indeholder hele den syntetiske CSF-DNA-sekvens. Efter ekspression og syrespalt-15 ning fås det samme CSF-derivat, som er'beskrevet i fig. 2.In FIG. 10 and the continuation; FIG. 10a, the preparation of the hybrid plasmids pS 200-204, which has synthetic CSF DNA sub-sequences, is shown, the plasmid pS 200 containing "Synthesis block I" of Annex I, the plasmid pS 201 10 containing "Synthetic block II" of Annex II, the plasmid pS 202 contains "Synthesis block III" according to Appendix III, the plasmid pS 203 contains the entire synthetic gene and pS 204 is an expression plasmid which also contains the entire synthetic CSF DNA sequence. After expression and acid cleavage, the same CSF derivative as described in FIG. 2nd
I fig. 11 og fortsættelsen, fig. 11a, deraf vises syntesen af ekspressionsplasmidet pS 207, som koder for et fusionsprotein, som efter spaltning med N-bromsuccinimid giver et CSF-derivat, i hvilket Trp i positionerne 13 og 20 122 hver gang er erstattet af His.In FIG. 11 and the continuation, FIG. 11a, there is shown the synthesis of the expression plasmid pS 207, which encodes a fusion protein which, after cleavage with N-bromosuccinimide, yields a CSF derivative in which Trp at positions 13 and 20122 is replaced each time by His.
I fig. 12 er vist en syntetisk DNA-delsekvens, som tillader fremstillingen af CSF-derivat, i hvilket Ile i position 100 er erstattet af Thr.In FIG. 12, a synthetic DNA part sequence is shown which allows the preparation of CSF derivative, in which Ile at position 100 is replaced by Thr.
I fig. 13 og fortsættelsen, fig. 13a, deraf vises 25 syntesen af ekspressionsplasmidet pS 210, som koder for et fusionsprotein, der efter cyanbromid-spaltning giver et CSF-derivat, i hvilket alle methionin-grupper er erstattet af neutrale aminosyrer, nemlig i stilling 36 af Ile og i stillingerne 46, 79 og 80 af Leu.In FIG. 13 and the continuation, FIG. 13a, there is shown the synthesis of the expression plasmid pS 210, which encodes a fusion protein which, upon cyanobromide cleavage, yields a CSF derivative in which all methionine groups are replaced by neutral amino acids, namely at position 36 of Ile and at positions 46 , 79 and 80 by Leu.
30 I fig. 14 vises en syntetisk DNA-sekvens, som ifølge synteseskemaet i fig. 13 tillader fremstillingen af et CSF-derivat, i hvilket Met i position 36 er erstattet af Ile og i position 46 af Leu, og i stedet for amino-syrerne 79 og 80 er der en eneste Leu-gruppe.In FIG. 14 shows a synthetic DNA sequence which according to the synthetic scheme of FIG. 13 allows the preparation of a CSF derivative in which Met at position 36 is replaced by Ile and at position 46 by Leu, and instead of amino acids 79 and 80 there is a single Leu group.
35 I fig. 15 vises til slut en syntetisk DNA, hvis anvendelse i synteseskemaet ifølge fig. 13 muliggør frem- 0 4 DK 170346 B1 stillingen af et CSF-derivat, i hvilket Mét i stilling 36 er erstattet af Ile og stilling 46 af Leu, og hvori de to aminosyrer i stilling 79 og 80 er fjernede. ♦35 In FIG. 15 finally shows a synthetic DNA whose use in the synthesis scheme of FIG. 13 enables the position of a CSF derivative in which Mét at position 36 is replaced by Ile and position 46 by Leu and wherein the two amino acids at positions 79 and 80 are removed. ♦
Det har vist sig, at den "åbne aflæsningsraster" 5 fra en DNA, som koder for interleukin-2, er især fordelagtig * som ekspressionshjælp til ekspression af peptider og proteiner, og at en N-terminal andel af IL-2, som i det væsentlige modsvarer de første hundrede aminosyrer, er særligt godt egnet' til fremstilling af fusionsproteiner.It has been found that the "open reading lattice" 5 of a DNA encoding interleukin-2 is particularly advantageous * as an expression aid for the expression of peptides and proteins, and that an N-terminal portion of IL-2, as in substantially equal to the first hundred amino acids, is particularly well suited for the production of fusion proteins.
10 Man får altså som hovedprodukt et fusionsprotéin, som fuldstændigt eller til den helt overvejende del består af eukaryotiske proteinsekvenser. På overraskende måde erkendes dette protein åbenbart ikke som fremmed protein af værts-proteaserne og nedbrydes heller ikke straks igen.Thus, as a major product, one obtains a fusion protein, which completely or to a large extent consists of eukaryotic protein sequences. Surprisingly, this protein is obviously not recognized as foreign protein by the host proteases and is not immediately degraded again.
15 Det er en yderligere fordel, at fusioiisproteinerne ifølge opfindelsen er tungtopløselige til uopløselige og følgelig" simpelt kan skilles fra de opløselige proteiner, hensigtsmæssigt ved hjælp af centrifugering. Da det ifølge opfindelsen i henseende til funktionen som "ballast-del" 20 af fusionsproteinet ikke kommer an på, at IL-2-delen er et biologisk aktivt molekyle, kommer det for så vidt heller ikke an på den eksakte struktur af IL-2-delen. Det er tilstrækkeligt, at i det væsentlige de hundrede første L-termi-nale aminosyrer foreligger. Det er altså eksempelvis muligt 25 at ved N-terminalen udføres variationer, som tillader en spaltning af fusionsproteinet, såfremt det ønskede protein er ordnet dertil N-terminalt. Omvendt kan man C-terminalt udføre variationer· for at muliggøre eller lette fraspaltningen af det ønskede protein.It is a further advantage that the fusion proteins of the invention are heavily soluble to insoluble and hence "simply separable from the soluble proteins, conveniently by centrifugation. because the IL-2 moiety is a biologically active molecule, it does not depend on the exact structure of the IL-2 moiety as well. For example, it is possible, for example, to carry out variations at the N-terminal which allow a cleavage of the fusion protein if the desired protein is arranged thereto N-terminally, Conversely, C-terminally, variations can be made to enable or facilitate the cleavage of the desired protein.
30 Den for human-IL-2 kodende naturlige DNA-sekvens er kendt fra EP-A nr 0.091.539. Den der anførte litteratur refererer til muse- og rotte-IL-2. Disse pattedyr-DNA kan tages i betragtning til syntesen af proteinerne ifølge opfindelsen. Hensigtsmæssigt går man imidlertid ud fra en 35 syntetisk DNA, især fordelagtigt fra en DNA for human-IL-2, som er beskrevet i det tyske offentliggørelsesskrift nr.The natural DNA sequence encoding human IL-2 is known from EP-A No. 0.091.539. The literature cited refers to mouse and rat IL-2. These mammalian DNAs may be considered for the synthesis of the proteins of the invention. Conveniently, however, one assumes a synthetic DNA, particularly advantageously from a human IL-2 DNA, which is disclosed in German publication specification no.
DK 170346 Bl 5 3.419.955 eller i EP-A 0.163.249. Denne syntetiske DNA har ikke kun det fortrin, at de i KODON-udvalget er afstemt til de givne forhold hos den hyppigst anvendte vært, E. coli, men den indeholder ligeledes en række spaltesteder 5 til restriktionsendonucleaser ved begyndelsen eller i om-*> rådet for den 100. triplet, hvilke man kan gøre brug af ifølge opfindelsen. Herved er det imidlertid ikke udelukket, at der i det mellemliggende område kan foretages variationer i DNA, hvorved man kan gøre brug af de yderligere 1° spaltesteder.DK 170346 Bl 5 3.419.955 or in EP-A 0.163.249. This synthetic DNA not only has the advantage of being matched in the KODON range to the conditions of the most frequently used host, E. coli, but it also contains a number of cleavage sites 5 for restriction endonucleases at the beginning or in the range for the 100th triplet which can be used according to the invention. However, this does not exclude the possibility that variations in the DNA can be made in the intermediate region, making use of the additional 1 ° gap sites.
Gør man brug af nucleaserne Ban II, Sac I eller Sst I, får man således en IL-2-delsekvens, som koder for ca. 95 aminosyrer. Denne længde er almindeligvis tilstrækkelig til at få et uopløseligt fusionsprotein. Hvis eksempel-15 vis et ønsket hydrofilt CSF-derivat alligevel ikke er tilstrækkeligt tungt opløselig^ men man ikke - for at producere så lidt "ballast" som muligt - men gøre brug af de ved C-terminalen nærliggende spaltesteder, kan man således •ed tilsvarende adapters eller linkers forlænge DNA-sekven-20 sen ved den N- og/eller C-terminale ende og således "spalte "ballast"-delen på mål". Man kan naturligvis også anvende DNA-sekvensen - mere eller mindre - til slutningen og således danne - eventuelt modificeret - biologisk aktivt IL-2 som "biprodukt".Thus, if you use the nucleases Ban II, Sac I or Sst I, you get an IL-2 sub-sequence which encodes approx. 95 amino acids. This length is usually sufficient to obtain an insoluble fusion protein. For example, if, for example, a desired hydrophilic CSF derivative is not sufficiently soluble, but one does not - to produce as little "ballast" as possible - but makes use of the cleft sites adjacent to the C-terminal, one can thus corresponding adapters or linkers extend the DNA sequence at the N- and / or C-terminal end, thus "cleaving the" ballast "portion on target". Of course, one can also use the DNA sequence - more or less - to the end, thus forming - possibly modified - biologically active IL-2 as a "by-product".
25 CSF-Molekylerne ifølge opfindelsen er ejendommelig ved, at de har den almene formel Pro-(As)X-CSF(12-126)-Z, hvori (As)x helt eller delvis betyder de første 11 aminosyrer i den naturlige CSF-sekvens, og Z betyder Glu eller Asp.The CSF molecules of the invention are characterized in that they have the general formula Pro- (As) X-CSF (12-126) -Z, wherein (As) x, in whole or in part, means the first 11 amino acids of the natural CSF- sequence and Z means Glu or Asp.
30 De omhandlede CSF-proteiner har det væsentlige kende tegn, at de ved N-terminalen indeholder aminosyren prolin.The CSF proteins at issue are essentially known to contain the amino acid proline at the N-terminal.
Disse CSF-proteiner forekommer i modsætning til naturligt forekommende humant GM-CSF ikke i det menneskelige legeme.In contrast to naturally occurring human GM-CSF, these CSF proteins do not occur in the human body.
Det skal i denne forbindelse fremhæves, at humant GM-CSF 35 naturligvis ikke virker antigent i det menneskelige legeme, da der ellers ville indtræde en autoimmunreaktion i legemet, 6 DK 170346 B1 hvilket da heller ikke er tilfældet hos sunde mennesker. På baggrund af artiklen af Benjamin et al. (1984) er det overraskende, at de omhandlede CSF-proteiner som ikke forekommer i naturen, ikke virker antigent, da Benjamin et al. allerede 5 har vist, at der dannes antistoffer mod høj konserverede proteiner, som kun afviger fra naturligt forekommende proteiner med hensyn til en eneste aminosyre. Det er netop på grund af de omhandlede CSF-proteiners manglende antigene potentiale, at det overhovedet er muligt at anvende disse 10 som farmaceutika til mennesker. I denne henseende er de endog det naturligt forekommende humane GM-CSF overlegne, da de er mere stabile overfor proteaser end human GM-CSF.In this connection, it should be emphasized that human GM-CSF 35 does not naturally act antigenically in the human body, as otherwise an autoimmune reaction would occur in the body, which is not the case in healthy humans either. Based on the article by Benjamin et al. (1984), it is surprising that the CSF proteins in question, which do not occur in nature, do not appear antigenic, as Benjamin et al. already 5 have shown that antibodies are formed against highly conserved proteins which differ only from naturally occurring proteins with respect to a single amino acid. It is precisely because of the lack of antigenic potential of the CSF proteins in question that it is at all possible to use these 10 as pharmaceuticals for humans. In this regard, they are even superior to the naturally occurring human GM-CSF, as they are more stable to proteases than human GM-CSF.
Fremgangsmåden ifølge opfindelsen er ejendommelig ved, at man indbygger et gen, der koder for CSF, i en bakte-15 riel ekspressionsvektor, transformerer"bakterier, især E. coli, dermed og bringer genet til ekspression deri.The method of the invention is characterized by incorporating a gene encoding CSF into a bacterial expression vector, thereby transforming bacteria, especially E. coli, into the gene for expression therein.
Spaltningen af fusionsproteinet kan ske på i og for sig kendt måde kemisk .‘eller enzymatisk. Valget af den egnede fremgangsmåde retter sig fremfor alt efter det 20 ønskede proteins aminosyresekvens. Er i broelementet Y ved carboxyterminalen tryptophan eller methionin eller Y Trp eller Met, kan der således ske en kemisk spaltning med N-bromsuccinimid eller halogencyan, såfremt der hver gang syntetiseres CSF-derivater, som ikke indeholder disse 2 5 amino syrer.The cleavage of the fusion protein can occur in a manner known per se, chemically or enzymatically. The choice of the appropriate method is primarily directed to the amino acid sequence of the desired protein. Thus, in the bridging element Y at the carboxy terminal tryptophan or methionine or Y Trp or Met, a chemical cleavage with N-bromosuccinimide or halogencyan can occur if CSF derivatives containing each of these 25 amino acids are synthesized each time.
CSF og derivater deraf, som i deres aminosyresekvens indeholder Asp-Pro og er tilstrækkeligt syrestabilt, kan, som det er allerede er vist ovenfor, proteolytisk spaltes på i og for sig kendt måde. Herved får man proteiner, 30 som indeholder N-terminalt prolin og C-terminalt asparaginsyre. P-å denne måde kan man altså også syntetisere modificerede proteiner.CSF and its derivatives, which in their amino acid sequence contain Asp-Pro and are sufficiently acid stable, can be proteolytically cleaved in a manner known per se, as already shown above. This gives proteins containing N-terminal proline and C-terminal aspartic acid. Thus, modified proteins can also be synthesized in this way.
Asp-Pro-bindingen kan udformes endnu mere syrelabilt, når dette broelement er (Asp)n~Pro eller 35 Glu-(Asp)n-Pro, idet n er 1-3.The Asp-Pro bond can be formed even more acid labile when this bridge element is (Asp) n ~ Pro or 35 Glu- (Asp) n-Pro, with n being 1-3.
7 DK 170346 B17 DK 170346 B1
Eksempler på enzymatiske spaltninger er ligeledes kendte, idet man også kan anvende modificerede anzymer med forbedret specificitet (jfr. C.S. Craik et al., Science 228 (1985) 291-297).Examples of enzymatic cleavages are also known in that modified enzymes with improved specificity can also be used (cf. C.S. Craik et al., Science 228 (1985) 291-297).
5 Fusionsproteinet udvindes ved ekspression i et bakterielt ekspressionssystem på i og for sig kendt måde.The fusion protein is recovered by expression in a bacterial expression system in a manner known per se.
Hertil egner alle kendte værts-vektorsystemer såsom bakterier af arterne Streptomyces, B. subtilis, Salmonella typhimurium eller Serratia marcescens, især E. coli, sig.To this end all known host vector systems such as bacteria of the species Streptomyces, B. subtilis, Salmonella typhimurium or Serratia marcescens, especially E. coli, are suitable.
10 DNA-sekvensen, som koder for det ønskede protein, indbygges på kendt måde i en vektor, som i det valgte ekspressionssystem sikrer en god ekspression.The DNA sequence encoding the desired protein is incorporated in a known manner into a vector which in the selected expression system ensures good expression.
I denne forbindelse vælger man hensigtsmæssigt promotoren og operatoren blandt trp, lac, tac, PL eller PR 15 fra fagen hsp, omp eller en syntetisk promotor, som de eksempelvis er foreslået i det tyske offentliggørelsesskrift nr. 3.430.683 eller i EP-A nr. 0.173.149. Fordelagtigt er tac-promotor-operator-sekvensen, som imidlertid er gængs i handelen (eksempelvis ekspressionsvektor pKK223-3, 20 Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, s. 63).In this connection, the promoter and operator are conveniently selected from trp, lac, tac, PL or PR 15 from the phage hsp, omp or a synthetic promoter, as they are, for example, proposed in German publication no. 3,430,683 or in EP-A no. 0.173.149. Advantageously, the tac promoter-operator sequence is commercially available (e.g., expression vector pKK223-3, Pharmacia, "Molecular Biologicals, Chemicals and Equipment for Molecular Biology", 1984, p. 63).
Ved ekspressionen af fusionsproteinet ifølge opfindelsen kan det vise sig at være hensigtsmæssigt at ændre enkelte tripletter i de første aminosyrer efter ATG-start-25 -codon for at hindre en eventuel basepardannelse på mRNA-planet. Sådanne ændringer som at fjerne eller addere enkelte aminosyrer er velkendte for fagfolk, og opfindelsen angår ligeledes disse ændringer.In the expression of the fusion protein of the invention, it may be appropriate to modify single triplets in the first amino acids after the ATG start-25 codon to prevent any base pair formation on the mRNA plane. Such changes as removing or adding single amino acids are well known to those skilled in the art, and the invention also relates to these changes.
Der foretrækkes CSF-proteiner, som i tilslutning til 30 det N-terminalt adderede prolin har hele CSF-aminosyresekven-sen. Det har dog overraskende vist sig, at også variationerne af CSF-molekylet, som fås ved eliminering af de første elleve aminosyrer, er biologisk aktive.CSF proteins are preferred which, in addition to the N-terminally added proline, have the entire CSF amino acid sequence. However, it has surprisingly been found that the variations of the CSF molecule obtained by elimination of the first eleven amino acids are also biologically active.
Fordelagtige er også varianter af opfindelsen, som 35 først leder til fusionsproteiner, som indeholder CSF-sekven-sen mere end én gang, fordelagtigt to eller tre gange. I dis- 0 8 DK 170346 B1 se fusionsproteiner er ballast-delen naturligt mindsket og udbyttet af det ønskede protein denned øges.Also advantageous are variants of the invention which first lead to fusion proteins containing the CSF sequence more than once, advantageously two or three times. In these fusion proteins, the ballast moiety is naturally diminished and the yield of the desired protein then increased.
Ved American Type Culture Cellection er plasmidet pHG 23 deponeret under nummeret ATCC 39900 i E. coli, og dette plasmid 5 fås ved indbygning af CSP-cDNA-sekvensen i Pst I-spaltestedet * "af pBR 322. DNA-sekvensen svarer herved til den i fig. 3 (B) af Wong et al. beskrevne variant. Ved indbygning gøres der brug af Pst I-spaltestedet nær 5'-enden på den ene side og af et ved GC-"Tailing" indført Pst I-sted ved 10 3'-enden (EP-A 0.183.350).At American Type Culture Cellection, the plasmid pHG 23 is deposited under the number ATCC 39900 in E. coli, and this plasmid 5 is obtained by incorporation of the CSP cDNA sequence into the Pst I cleavage site * "of pBR 322. The DNA sequence corresponds to the The variant described in Fig. 3 (B) by Wong et al., for incorporation, uses the Pst I cleavage site near the 5 'end on one side and a Pst I site introduced by GC "Tailing" at 10 3 'end (EP-A 0.183.350).
Eksempel 1 Pro°-CSFExample 1 Pro ° -CSF
(5 Vektoren pUC 12 åbnes med Eco' RI og Pst I, og det store fragment (16) isoleres. Dette fragment (16) ligeres med det syntetiske DNA-fragment (17) og fragmentet (2) (eksempel 1? figur 1). Kompetente celler af E. coli JM 103 transformeres med ligeringsblandingen, og man selekterer 20 de ønskede kloner, som indeholder plasmidet pW 212 (18).(5 The vector pUC 12 is opened with Eco 'RI and Pst I and the large fragment (16) is isolated. This fragment (16) is ligated with the synthetic DNA fragment (17) and fragment (2) (Example 1? Figure 1) Competent cells of E. coli JM 103 are transformed with the ligation mixture, and 20 of the desired clones containing the plasmid pW 212 (18) are selected.
Fra plasmid-DNA fraspaltes fragmentet (19) med Pvu I og Pst I, og dette fragment indeholder CSF--sekvensen.From plasmid DNA, fragment (19) is cleaved with Pvu I and Pst I, and this fragment contains the CSF sequence.
Ved indføjelse af lac-repressoren (P.J. Parabaugh, 25 Nature 274 (1978) 765-769) i plasmidet pKK 177-3 med pUC 8-polylinkeren (Amann et al., Gene 25 (1983) 167? EP-A nr. 0.133.282) får man plasmidet pJF118 (20) (fig. 1; jfr. tysk patentansøgning nr. P 3.526.995.2, eksempel 6, fig. 6). Dette åbnes ved det singulære restriktionssted 30 for Ava I og formindskes på i og for sig kendt måde ved exonuclease-behandling til ca. 1000 bp. Efter ligering fås plasmidet pEW 1000 (21), hvori man får lac-repres-sorgenet fuldstændigt, som imidlertid på grund af formindskelsen foreligger i tydeligt større antal kopier end 35 udgangsplasmidet.By inserting the lac repressor (PJ Parabaugh, 25 Nature 274 (1978) 765-769) into plasmid pKK 177-3 with the pUC 8 polylinker (Amann et al., Gene 25 (1983) 167? EP-A No. 0.133 .282) gives the plasmid pJF118 (20) (Fig. 1; cf. German Patent Application No. P 3,526,995.2, Example 6, Fig. 6). This is opened at the singular restriction site 30 of Ava I and is reduced in a manner known per se by exonuclease treatment to approx. 1000 bp. After ligation, the plasmid pEW 1000 (21) is obtained, whereby the lac reporter gene is completely obtained, which, however, due to the decrease, has a significantly larger number of copies than the starting plasmid.
DK 170346 B1 0 ' 9 I stedet for plasmidet pKK177-3 kan man ligeledes udgå fra det ovennævnte i handelen gængse plasmid pKK223-3, indbygge lac-repressoren og forkorte det frembragte produkt analogt.Instead of the plasmid pKK177-3, the above-mentioned commercially available plasmid pKK223-3 can also be used, incorporate the lac repressor and shorten the produced product by analogy.
5 Plasmidet pEW 1000 (21) åbnes med restriktions enzymerne EcoR I og Sal I, og fragmentet (22) isoleres.The plasmid pEW 1000 (21) is opened with the restriction enzymes EcoR I and Sal I and the fragment (22) is isolated.
Plasmidet pl59/6 (23), fremstillet ifølge tysk offentliggørelsesskrift nr. 3.419.995 (EP-A nr. 0.163.249), eksempel 4 (fig. 5), åbnes med restriktionsenzymerne 10 Eco RI og Sal I, og det lille fragment (24) isoleres, og dette fragment indeholder IL-2-sekvensen.Plasmid pl59 / 6 (23), prepared according to German Publication No. 3,419,995 (EP-A No. 0.163,249), Example 4 (Fig. 5), is opened with the restriction enzymes 10 Eco RI and Sal I, and the small fragment (24) is isolated and this fragment contains the IL-2 sequence.
Ved ligering af fragmenterne(22) og (24) fås hybridplasmidet pEW 1001 (25).By ligating the fragments (22) and (24), the hybrid plasmid pEW 1001 (25) is obtained.
Plasmidet (25) åbnes på den ene side med Eco RI 15 og Pvu I, hvorved man får fragment (26) , hvilket indeholder den største del af IL-2-sekvensen. Denne delsekvens betegnes i figurerne med "/\IL2".The plasmid (25), on the one hand, is opened with Eco RI 15 and Pvu I to give fragment (26), which contains the major part of the IL-2 sequence. This part sequence is denoted in the figures by "/ \ IL2".
På den anden side åbnes plasmidet (25) med Eco RI og Pst I, og det store fragment (27) isoleres.On the other hand, the plasmid (25) is opened with Eco RI and Pst I and the large fragment (27) is isolated.
20 Ved ligering af fragmenterne (19), (26) og (27), transformation af kompetente E. coli 294-celler og selektion får kloner, som indeholder plasmidet pW 216 (28). Plasmid-DNA karakteriseres ved restriktions- og DNA--sekvensanalyse.By ligating the fragments (19), (26) and (27), transformation of competent E. coli 294 cells and selection yield clones containing the plasmid pW 216 (28). Plasmid DNA is characterized by restriction and DNA sequence analysis.
25 En kultur af E. coli-celler, der står natten over, og som indeholder plasmidet (28), fortyndes med LB-medium (J. H. Miller, se ovenfor), der indeholder 50 ^ig/ml ampi-cillin, i et forhold på ca. 1:100, og væksten følges via OD-måling. Ved OD = 0,5 indstilles kulturen til 30 1 mM isopropyl-3-galactopyranosid (IPTG), og bakterierne centrifugeres fra efter 150-180 minutters forløb. Bakterierne koges i 5 minutter i en pufferblanding (7 molær urinstof, 0,1% SDS, 0,1 molær natriumphosphat, pH-værdi = 7,0), og der påføres prøver på en SDS-gelelektroforeseplade.A culture of overnight E. coli cells containing the plasmid (28) is diluted with LB medium (JH Miller, see above) containing 50 µg / ml ampicillin at a ratio of approx. 1: 100, and growth is monitored via OD measurement. At OD = 0.5, culture is adjusted to 30 1 mM isopropyl-3-galactopyranoside (IPTG) and the bacteria are centrifuged after 150-180 minutes. The bacteria are boiled for 5 minutes in a buffer mixture (7 molar urea, 0.1% SDS, 0.1 molar sodium phosphate, pH = 7.0) and samples are applied to an SDS gel electrophoresis plate.
35 Efter elektroforese fås et proteinbånd fra bakterier, som indeholder plasmidet (28), og dette bånd svarer til o 10 DK 170346 B1 størrelsen af det forventede fusionsprotein. Efter opluk-ning af bakterierne (French Press; Dyno-Miihle) og centrifugering befinder fusionsproteinet sig i udfældningen/ således at allerede i den ovenstående væske betydelige mængder af 5 de øvrige proteiner kan skilles fra. Efter isolering af * ‘fusionsproteinet frigøres det forventede CSF-derivat, som indeholder yderligere N-terminalt prolin/ ved syrespaltning. Dette viser aktivitet i den biologiske test.After electrophoresis, a protein band is obtained from bacteria containing the plasmid (28) and this band corresponds to the size of the expected fusion protein. After picking up the bacteria (French Press; Dyno-Miihle) and centrifuging, the fusion protein is in the precipitate / so that already in the above liquid considerable amounts of the other proteins can be separated. After isolation of the fusion protein, the expected CSF derivative containing additional N-terminal proline / by acid cleavage is released. This shows activity in the biological test.
De angivne induktionsbetingelser gælder for ryste-10 kulturer; ved større fermenteringer er det hensigtsmæssigt med tilsvarende ændrede OD-værdier og eventuelt let varierede IPTG-koncentrationer.The indicated induction conditions apply to shaking cultures; For larger fermentations, correspondingly altered OD values and possibly slightly different IPTG concentrations are appropriate.
Eksempel 2 15 Pro1-CSF(2-127)Example 2 Pro1-CSF (2-127)
Ved ligering af fragmenterne (2) (fig. 1) og (16) (fig. 2) med den syntetiske DNA-sekvens (29) får man hybrid-plasmidet (30), som lige med undtagelse af den syntetiske DNA-sekvens modsvarer plasmidet (18).By ligating the fragments (2) (Fig. 1) and (16) (Fig. 2) with the synthetic DNA sequence (29), the hybrid plasmid (30) is obtained which corresponds equally with the exception of the synthetic DNA sequence. the plasmid (18).
20 Fra plasmidet (30) fraspaltes med Pvu I og Pst IThe plasmid (30) is cleaved with Pvu I and Pst I
fragmentet (31), som indeholder CSF-DNA-sekvensen, hvori codon for den første aminosyre imidlertid er erstattet af en codon for protein. Ved ligering af fragmentet (31) ved fragmenterne (26) og (27) får man hybridplasmidet 25 pW 240 (32). Ekspressionen i E. coli, som gennemføres ifølge eksempel 1/ giver et CSF-derivat, hvori den første aminosyre er erstattet af prolin. Også dette derivat har biologisk aktivitet.however, the fragment (31) containing the CSF DNA sequence in which the codon for the first amino acid is replaced by a codon for protein. By ligating the fragment (31) to fragments (26) and (27), the hybrid plasmid 25 pW 240 (32) is obtained. The expression in E. coli carried out according to Example 1 gives a CSF derivative in which the first amino acid is replaced by proline. This derivative also has biological activity.
30 Eksempel 3 CSF(2-127)Example 3 CSF (2-127)
Et plasmid, som indeholder CSF-DNA-sekvensen med et Pst-I-restriktionssted på 3'-enden deraf, eksempelvis plasmidet pHG 23 (ATCC 39900) , spaltes med Sfa NI,A plasmid containing the CSF DNA sequence with a Pst-I restriction site at the 3 'end thereof, for example plasmid pHG 23 (ATCC 39900), is digested with Sfa NI,
35 og det lineariserede plasmid (34) opfyldes delvist ved hjælp af Klenow-polymerase og GTP. Det udragende nucleotid A35 and the linearized plasmid (34) is partially fulfilled by Klenow polymerase and GTP. The protruding nucleotide A
o 11 DK 170346 B1o 11 DK 170346 B1
Fra plastxnidet (43) isoleres fragmentet (44) med Pvu I og Pst I, og dette fragment indeholder DNA--sekvensen for CSF-derivatet. Dette fragment (44) ligeres med fragmenterne (26) og (27), hvilket leder til dannelsen 5 af hybridplasmidet pW 242 (45).From the plastic cut (43), the fragment (44) is isolated with Pvu I and Pst I, and this fragment contains the DNA sequence of the CSF derivative. This fragment (44) is ligated to fragments (26) and (27), leading to the formation 5 of the hybrid plasmid pW 242 (45).
Ekspressionen ifølge eksempel 1 leder til dannelsen af et fusionsprotein, ud fra hvilket man efter syrespaltning får et CSF-derivat, i hvilket de første fem aminosyrer mangler. Også dette produkt er biologisk aktivt.The expression of Example 1 leads to the formation of a fusion protein from which, after acid cleavage, a CSF derivative is obtained in which the first five amino acids are missing. This product is also biologically active.
10 fraspaltes med Sl-nuclease og derpå fraspaltes fragmentet (35) med Pst I.10 is cleaved with S1 nuclease and then the fragment (35) is cleaved with Pst I.
Ved ligering af fragmentet (35) med den syntetiske DNA-sekvens (36) og fragmentet (16) (fig. 2) får man plasmi-det (37), som er analogt til plasmidet (18).By ligating the fragment (35) with the synthetic DNA sequence (36) and the fragment (16) (Fig. 2), the plasmid (37) analogous to the plasmid (18) is obtained.
15 Fra plasmidet (37) fraspaltes fragmentet (38) ved hjælp af Pvu I og Pst I. Dette fragment ligeres med fragmenterne (26) og (27), hvorved man får plasmidet pW 241 (39) .From the plasmid (37), the fragment (38) is cleaved by Pvu I and Pst I. This fragment is ligated with the fragments (26) and (27) to give the plasmid pW 241 (39).
Ved ekspression ifølge eksempel 1- får man fusions-20 protein, som efter syrespaltning giver et CSF-derivat, hvori den første aminosyre mangler. Dette derivat er biologisk aktivt.By expression of Example 1, fusion protein is obtained, which after acid cleavage gives a CSF derivative in which the first amino acid is missing. This derivative is biologically active.
Eksempel 4 25 CSF (6-127).Example 4 CSF (6-127).
Plasmidet (33) (eller et tilsvarende plasmid, som indeholder CSF-*-DNA-sekvensen) spaltes først helt med Pst I og derpå delvist med Bst NI, og fragmentet (40) isoleres.The plasmid (33) (or a similar plasmid containing the CSF - * DNA sequence) is first cleaved completely with Pst I and then partially with Bst NI and the fragment (40) isolated.
De syntetiske DNA-sekvenser (41) og (36) (fig. 4) 30 ligeres først til sekvensen (42), og denne ligeres derpå med fragmentet (16) (fig. 2) og fragmentet (40) , hvorved man får plasmidet pW 212 (43).The synthetic DNA sequences (41) and (36) (Fig. 4) are first ligated to the sequence (42) and then ligated to the fragment (16) (Fig. 2) and the fragment (40) to obtain the plasmid. pW 212 (43).
35 0 12 DK 170346 B135 0 12 DK 170346 B1
Eksempel 5 CSF(8-127).Example 5 CSF (8-127).
Ligerer man først den syntetiske DNA-sekvens (36) (fig. 4) med den syntetiske DNA-sekvens (46) og derpå 5 det således tilvejebragte DNA-fragment (47) med fragmenterne (40) og (16), får man således hybridplasmidet (48).If one first compares the synthetic DNA sequence (36) (Fig. 4) with the synthetic DNA sequence (46) and then the DNA fragment (47) thus obtained with the fragments (40) and (16) is thus obtained. the hybrid plasmid (48).
Fra dette spaltes ved hjælp af Pvu I og Pst II fragmentet (49) , som indeholder DNA-sekvensen for CSF-derivatet.From this, the Pvu I and Pst II fragment (49), which contains the DNA sequence of the CSF derivative, is cleaved.
Ligering af fragmenterne (49, (26) og (27) giver hybrid-10 plasmidet pW 243 (50), som svarer til plasmidet (45) lige med undtagelse af den forkortede DNA-sekvens for CSF-derivatet.Ligation of fragments (49, (26) and (27) yields the hybrid 10 plasmid pW 243 (50) which corresponds to the plasmid (45) equally except for the abbreviated DNA sequence of the CSF derivative.
Ekspressionen ifølge eksempel 1 leder til et fusionsprotein, som efter syrespaltning giver et CSF-deri-15 vat, hvori de første syv aminosyrer mångler. Også dette derivat er biologisk aktivt.The expression of Example 1 leads to a fusion protein which, after acid cleavage, yields a CSF derivative in which the first seven amino acids are lacking. This derivative is also biologically active.
Eksempel 6 CSF(12-127).Example 6 CSF (12-127).
20 Ligerer man den syntetiske DNA-sekvens (51) med fragmenterne (33) og (16), får man således hybridplasmidet (52). Spalter man fra dette ved hjælp af Pvu I og Pst I sekvensen (53), som indeholder DNA-sekvensen for CSF-derivatet, og ligerer dette fragment (53) med fragmenterne (26) 25 og (27), får man således hybridplasmidet pW 244 (54), der svarer til plasmidet (45) lige med undtagelse af den forkortede CSF-sekvens.If one synthesizes the synthetic DNA sequence (51) with the fragments (33) and (16), the hybrid plasmid (52) is thus obtained. If this is cleaved by the Pvu I and Pst I sequence (53) containing the DNA sequence of the CSF derivative and ligated this fragment (53) with the fragments (26) 25 and (27), the hybrid plasmid pW is thus obtained 244 (54) corresponding to the plasmid (45) equally except for the abbreviated CSF sequence.
Ekspressionen ifølge eksempel 1 leder til et fusionsprotein, som efter syrespaltning giver et CSF-derivat, 30 hvori aminosyrerne 1-11 er fjernede. Også dette forkortede molekyle er biologisk aktivt.The expression of Example 1 leads to a fusion protein which, after acid cleavage, yields a CSF derivative in which amino acids 1-11 are removed. This abbreviated molecule is also biologically active.
Eksempel 7 -Example 7 -
Pro°-CSF(1-126)-Asp.Pro ° -CSF (1-126) -Asp.
35 Man spalter delvist DNA-sekvensen (19) (fig. 2) med Bst NI og isolerer fragmentet (55), som indeholder o 13 DK 170346 B1 den største del CSF-sekvensen.35 The DNA sequence (19) (Fig. 2) is partially cleaved with Bst NI and the fragment (55) containing the major portion of the CSF sequence is isolated.
Ved spaltning af plasmidet (33) (fig. 4) (eller et tilsvarende plasmid, som indeholder CSF-DNA-sekvensen) med først Pst I og derpå partielt med Bst NI får man DNA-5 -sekvensen (56), som omfatter den største del af CSF--sekvensen.Upon cleavage of the plasmid (33) (Fig. 4) (or a similar plasmid containing the CSF DNA sequence) with first Pst I and then partially with Bst NI, the DNA-5 sequence (56) comprising the most of the CSF sequence.
Man syntetiserer DNA-sekvensen (57) , som komplete-rer sekvensen (56) til en DNA-sekvens, som koder for CSF--derivat, i hvis C-terminal glutaminsyren er erstattet'af 1o asparaginsyre.The DNA sequence (57), which completes the sequence (56), is synthesized into a DNA sequence encoding CSF derivative in which the C-terminal glutamic acid is replaced by 1o aspartic acid.
Vektoren pUC 13 åbnes med Pst I og Sma I og man isolerer det store fragment (58). Ligerer man dette restplasmid (58) med fragmenterne (56) og (57)^ får man således hybrid-plasmidet pW 245 (59) med den C-terminalt modificerede 15 sekvens.The vector pUC 13 is opened with Pst I and Sma I, isolating the large fragment (58). Thus, if this residual plasmid (58) is compared with fragments (56) and (57), the hybrid plasmid pW 245 (59) is obtained with the C-terminally modified sequence.
Fra plasmidet (59) spalter man ved hjælp af Sfa NI og Pst I fragmentet (60) fra, som indeholder den modificerede CSF-DNA-sekvens. Dette fragment (60) ligeres med den syntetiske DNA-sekvens (61) og fragmentet 20 (55), hvorved man får DNA-sekvensen (62). Denne ligeres med DNA-fragmenterne (26) og (27) (fig. 2), hvorved man får hybridplasmidet pw 246 (63). Dette plasmid er gengivet to gange i'fig. 8a, idet man skal hente det kodende fusionsproteins aminosyresekvens fra den nederste gengivelse.From the plasmid (59) the fragment (60) containing the modified CSF DNA sequence is cleaved by Sfa NI and Pst I. This fragment (60) is ligated to the synthetic DNA sequence (61) and fragment 20 (55) to give the DNA sequence (62). This is ligated to the DNA fragments (26) and (27) (Fig. 2) to give the hybrid plasmid pw 246 (63). This plasmid is reproduced twice in FIG. 8a, taking the amino acid sequence of the coding fusion protein from the lower representation.
25 Ved ekspressionen ifølge eksempel 1 fås et fusions protein, af hvilket der efter syrespaltning fremkommer et CSF-derivat, som N-terminalt er forlænget med prolin, og hvori den sidste aminosyre desuden er erstattet af asparaginsyre. Dette derivat er biologisk aktivt.By the expression of Example 1, a fusion protein is obtained, of which, after acid cleavage, a CSF derivative is obtained which is N-terminally extended with proline and in which the last amino acid is also replaced by aspartic acid. This derivative is biologically active.
3030
Eksempel 8 Pro°-CSF(1-126)-AspExample 8 Pro ° -CSF (1-126) -Asp
Man spalter hybridplasmidet (63) (fig. 8) med Eco RI og Pst I og isolerer fragmentet, som i 35 tilslutning til IL-2-delsekvensen indeholder de to modificerede CSF-sekvenser. Denne sekvens (64) spaltes DK 170346 Bl 14The hybrid plasmid (63) (Fig. 8) is cleaved with Eco RI and Pst I and isolated the fragment which, in association with the IL-2 sub sequence, contains the two modified CSF sequences. This sequence (64) is cleaved DK 170346 B1 14
OISLAND
delvis med Rsa I, og de to fragmenter (65) og (66) isole res. Ved ligering af DNA-sekvenserne (27), (65), (67), (61) og (60) får man hybridplasmidet pW 247 (68), hvori de ligerede sekvenser er ordnede i den nævnte rækkefølge.partially with Rsa I, and the two fragments (65) and (66) are isolated. By ligating the DNA sequences (27), (65), (67), (61) and (60), the hybrid plasmid pW 247 (68) is obtained in which the ligated sequences are arranged in the order mentioned.
5 Ekspressionen ifølge eksempel 1 giver et fusions- protein, ud fra hvilket der efter syrespaltning fås det samme CSF-derivat som ifølge eksempel 7.The expression of Example 1 gives a fusion protein from which, after acid cleavage, the same CSF derivative as of Example 7 is obtained.
Eksempel 9 10 Syntetisk gen (for Pro°-CSF).Example 9 Synthetic gene (for Pro ° -CSF).
Ved i og for sig kendte fremgangsmåder, eksempelvis ifølge phosphitmetoden (tysk offentliggørelsesskrift nr. 3.327.007, 3.328.793, 3.409.966, 3.414.831 og 3.419.995) syntetiseres de tre "synteseblbkke"I (CSF-I), i figurerne .jg betegnet som (69) , II (CAF-II)j i figurerne som (70) , og III (CSF-III); i figurerne som (71) . I disse synteseblokkes nucleotidfølge er de syntetiserede oligonucleotider Ia-Im, Ila-IIf og Illa-IIIl indtegnede (bilag).By methods known per se, for example, according to the phosphite method (German Publication No. 3,327,007, 3,328,793, 3,409,966, 3,414,831 and 3,419,995), the three "synthesis blocks" I (CSF-I) are synthesized in Figures .jg designated as (69), II (CAF-II) j in Figures as (70), and III (CSF-III); in the figures as (71). In the nucleotide sequence of these synthesis blocks, the synthesized oligonucleotides Ia-Im, Ila-IIf and Illa-IIIl are plotted (appendix).
Med valget af nucleotiderne til det syntetiske 20 gen fastsættes der ikke kun singulære spaltesteder ved de tre synteseblokkes sammenstykningssteder, men inden for genfragmenterne ligeledes en række singulære restriktionssteder. Disse er angivne i de følgende tabeller. Disse singulære restriktionssteder kan på i og for sig kendt måde 25 tjene til at udskifte, at supplere eller fjerne codons for aminosyrer.With the choice of the nucleotides for the synthetic gene, not only are singular cleavage sites determined at the junction sites of the three synthesis blocks, but within the gene fragments also a number of singular restriction sites. These are listed in the following tables. These singular restriction sites may, in a manner known per se, serve to replace, supplement or remove amino acid codons.
30 35 0 15 DK 170346 B130 35 0 15 DK 170346 B1
Sgiteseblok I (CSF I)_ Spaltning efter nucleotid nr.Site block I (CSF I) - Cleavage by nucleotide no.
Enzym Genkendelsessekvens (kodende streng)Enzyme Recognition Sequence (coding strand)
Nar I GG+CGCC 1 5 Hpa II C+CGG 4 ‘ Hae II GGCGC4C 4When I GG + CGCC 1 5 Hpa II C + CGG 4 'Hae II GGCGC4C 4
Nae I GCC4GGC 5Nae I GCC4GGC 5
Pvu I CGAT+CG 13Pvu I CGAT + CG 13
Sal I G+TCGAC 24 10 Acc I GT4CGAC 25Will I G + TCGAC 24 10 Acc I GT4CGAC 25
Hine II GTC4GAC 26Hine II GTC4GAC 26
Hpa 1/ GTT4AAC 48Hpa 1 / GTT4AAC 48
Hine IIHine II
Hha I GCG40 66 15 Hinf I G+AGTC \ 88Hha I GCG40 66 15 Hinf I G + AGTC \ 88
Nru I TCG+CGA 89Nru I TCG + CGA 89
Xma III ClGGCCG 95Xma III ClGGCCG 95
Sac II CCGC4CG 101Sac II CCGC4CG 101
Eco RV GAT4ATC 128 20Eco RV GAT4ATC 128 20
Synteseblok II (CSF-II) 25 Spaltning efter nucleotid nr.Synthesis Block II (CSF-II) Cleavage by Nucleotide no.
Enzym Genkendelsessekvens (kodende streng)Enzyme Recognition Sequence (coding strand)
Afl III A4CATGT 157Afl III A4CATGT 157
Mlu I A4CGCGT 169 30 Xho I ClTCGAC 175Mlu I A4CGCGT 169 30 Xho I ClTCGAC 175
Tag I T+CGA 176Take I T + CGA 176
Hga I GACGC (5/10) 177Hga I GACGC (5/10) 177
Ava I C+TCGAG 177Ava I C + TCGAG 177
Alu I AG+CT 180 35 Sac 1/ GAGCT+C 182Alu I AG + CT 180 35 Sac 1 / GAGCT + C 182
Hgi AIHgi AI
Stu 1/ AGG+CCT 194Stu 1 / AGG + CCT 194
Hae IHae I
0 16 DK 170346 B10 16 DK 170346 B1
Synteseblok III (CSF-III)Synthetic Block III (CSF-III)
Spaltning efter nucleotid nr. Enzym Genkendelsessekvens (kodende streng) 5 Afl II C+TTAAG 217 - Hae III GG+CC 224Cleavage by Nucleotide No. Enzyme Recognition Sequence (coding strand) 5 Breed II C + TTAAG 217 - Hae III GG + CC 224
Apa I GGGCC+C 227Monkey I GGGCC + C 227
Mnl I CCTC (7/7) 238Mnl I CCTC (7/7) 238
Nhe I G*CTAGC 241 10 Mae I C+TAG 242Nhe I G * CTAGC 241 10 Mae I C + TAG 242
Aha II GA+CGTC 280Aha II GA + CGTC 280
Aat II GACGT4C 283Aat II GACGT4C 283
Sci NI G4CGC 287Sci NI G4CGC 287
Mst I TCG+GCA 288 15 Sau 3AI/ IGATC 296Mst I TCG + GCA 288 15 Sau 3AI / IGATC 296
Mb o IMb o I
Dpn I GA+TC 298Dpn I GA + TC 298
Asu II TT+CGAA 308Asu II TT + CGAA 308
Aha III TTT+AAA 318 20 Ava II G4GTCC 382Aha III TTT + AAA 318 20 Ava II G4GTCC 382
Eco RI I +CCAGG 384Eco RI I + CCAGG 384
Bst NI/ CC+AGG 380Bst NI / CC + AGG 380
Ser PISer PI
25 De tre synteseblokke klones først enkeltvis, forøges i E. coli og isoleres igen:The three synthesis blocks are first cloned individually, increased in E. coli and isolated again:
Synteseblokken CSF-I (69) indbygges i pUC 12-derivatet (16), hvorved man får plasmidet pS 200 (72).The synthesis block CSF-I (69) is incorporated into the pUC 12 derivative (16) to give the plasmid pS 200 (72).
pUC 12 åbnes med restriktionsenzymerne Pst I og 30 Hind III og ligeres med det lineariserede plasmid (73) fra synteseblokken CSF-II (70), hvorved man får plasmidet pS 201 (74) .pUC 12 is opened with the restriction enzymes Pst I and 30 Hind III and ligated with the linearized plasmid (73) from the synthesis block CSF-II (70) to give the plasmid pS 201 (74).
pUC 13 åbnes med Hind III og Sma I, og det linea-riserede plasmid (75) ligeres med CSF-III, hvorved man får 35 plasmidet pS 202 (76).pUC 13 is opened with Hind III and Sma I, and the linearized plasmid (75) is ligated with CSF-III to give the plasmid pS 202 (76).
0 17 DK 170346 B10 17 DK 170346 B1
De reisolerede synteseblokke (69)/ (70) og (71) ligeres derpå i den med Eco RI og Sma II lineariserede vektor pUC 12 (77), idet man får plasmidet pS 203 (78).The reinsulated synthesis blocks (69) / (70) and (71) are then ligated into the Eco-RI and Sma II linearized vector pUC 12 (77) to obtain plasmid pS 203 (78).
Dette hybridplasmid forøges - såsom plasmiderne med de en-5 kelte synteseblokke - i E. coli 79/02, og det syntetiske gen karakteriseres ved restriktions- og sekvensanalyse.This hybrid plasmid is increased - such as the plasmids with the single synthesis blocks - in E. coli 79/02, and the synthetic gene is characterized by restriction and sequence analysis.
Plasmidet (78) spaltes partielt med Pvu I og Barn HI, og det lille fragment (79) isoleres med hele CSF-sekvensen.The plasmid (78) is partially cleaved with Pvu I and Barn HI, and the small fragment (79) is isolated with the entire CSF sequence.
10 Ekspressionsplasmidet (21) åbnes med Eco RI ogThe expression plasmid (21) is opened with Eco RI and
Barn HI, og det store fragment (80) isoleres. Dette fragment (80) ligeres derpå med fragmentet (26), som indeholder IL-2-delsekvensen, og det syntetiske gen (79). Herved får man plasmidet pS 204 (81) som koder for et fusionsprotein, 15 i hvilket der på IL-2-delsekvensen først følger broelementet, som tillader syrespaltningen, og derpå aminosyresekven-sen af CSF. Ved syrespaltning fås følgelig et CSF-derivat, som er N-terminalt forlænget med prolin.Child HI, and the large fragment (80) is isolated. This fragment (80) is then ligated to the fragment (26) containing the IL-2 sub sequence and the synthetic gene (79). Thereby the plasmid pS 204 (81), which encodes a fusion protein, is obtained, in which, on the IL-2 sub sequence, the bridge element which allows the acid cleavage is followed first and then the amino acid sequence of CSF. Accordingly, by acid cleavage a CSF derivative is obtained which is N-terminally extended by proline.
20 Eksempel 10 CSF(1-12)His(14-121)His(123-127)Example 10 CSF (1-12) His (14-121) His (123-127)
Erstatter man i synteseblok I oligonucleotiderne la og Ib samt Ic og Id med de syntetiske sekvenser (82) og (83), får man således en modificeret synteseblok I, som ko- ..Substituting in synthesis block I oligonucleotides 1a and 1b as well as Ic and 1d with the synthetic sequences (82) and (83), a modified synthesis block I is thus obtained as co ..
25 der for en CSF-I-analog, hvori Trp står foran den første aminosyre (Ala) og i position 13 erstattes Trp med His.25 for a CSF-I analog in which Trp is in front of the first amino acid (Ala) and at position 13, Trp is replaced by His.
Plasmidet (72) (fig. 10) åbnes med Eco RI og Hpa I, og det store fragment (84) isoleres. Dette ligeres derpå med de syntetiske fragmenter (82) og (83), hvorved man får 30 plasmidet pS 205 (85) som koder for dette modificerede CSF I- (CSF-11) .The plasmid (72) (Fig. 10) is opened with Eco RI and Hpa I, and the large fragment (84) is isolated. This is then ligated to the synthetic fragments (82) and (83) to give the plasmid pS 205 (85) encoding this modified CSF I- (CSF-11).
Man åbner plasmidet (76) (fig. 10) med Hind III og Sal I og isolerer det lille (86) og det store (87) fragment. Det lille fragment (86) efterspaltes med Taq I} ogThe plasmid (76) (Fig. 10) is opened with Hind III and Sal I and the small (86) and large (87) fragment isolated. The small fragment (86) is cleaved with Taq I} and
OCOC
fragmentet (88) isoleres.the fragment (88) is isolated.
Det store fragment (87) ligeres derpå med (88) og ' 18 DK 170346 B1 0 det syntetiske fragment (89), i hvilket codon for Trp i position 122 er erstattet med His, idet man får plasmidet pS 206 (90), som koder for det modificerede CSF III (CSF-III')· Dette plasmid transformeres, forøges og re-5 isoleres efter E. coli, spaltes med Hind III og Sal I, “ og det lille fragment (91), som koder for CSF-III', isoleres.The large fragment (87) is then ligated with (88) and the synthetic fragment (89) in which the codon for Trp at position 122 is replaced with His, yielding the plasmid pS 206 (90), which encoding the modified CSF III (CSF-III ') · This plasmid is transformed, amplified and re-isolated after E. coli, cleaved with Hind III and Sal I,' and the small fragment (91) encoding CSF III ', isolated.
Plasmidet (85) spaltes partielt med Pvu I og med Pst I, og det lille fragment (92), som koder for CSF-I' isoleres.The plasmid (85) is partially cleaved with Pvu I and with Pst I, and the small fragment (92) encoding CSF-I 'is isolated.
10 Ligerer man derpå fragmenterne (22), (26), (92), (70) og (91), får man således plasmidet pS 207 (93). Dette koder for et fusionsprotein, i hvilket der på IL-2-delse-kvensen følger et broelement, som umiddelbart foran den første aminosyre af CSF (Ala) indeholder Trp. Da Trp i CSF-15 -molekylet i positionerne 13 og 122 erstattes af His kan der derefter ske en spaltning af fusionsproteinet med N-brom-succinimid. Derved får man CSF--derivatet, hvori tryptophan i begge positioner er erstattet af histidin.If fragments (22), (26), (92), (70) and (91) are then ligated, the plasmid pS 207 (93) is thus obtained. This encodes a fusion protein in which on the IL-2 partition sequence follows a bridge element which immediately in front of the first amino acid of CSF (Ala) contains Trp. Since Trp in the CSF-15 molecule at positions 13 and 122 is replaced by His, the fusion protein can be cleaved with N-bromo-succinimide. This gives the CSF derivative, in which tryptophan in both positions is replaced by histidine.
20 Eksempel 11 CSF(1-99)Thr(101-127)Example 11 CSF (1-99) Thr (101-127)
Erstatter man ved syntesen af synteseblokken III oligonucleotiderne Ille og Ulf med den syntetiske sekvens (94); og går man i øvrigt frem ifølge eksempel 9, får man 25 således et CSF-derivat, hvori ILe i position 100 er erstatte't af Thr.Replacing by the synthesis of the synthesis block III the oligonucleotides Ille and Ulf with the synthetic sequence (94); and if otherwise proceeded according to Example 9, there is thus obtained a CSF derivative in which ILe at position 100 is replaced by Thr.
Eksempel 12 CSF(1-35)Ile(37-45)Leu(47-78)Leu-Leu(81-127) 30 Man syntetiserer først oligonucleotidet (95), som i position 36 indeholder codon for Ile i stedet for Met, og oligonucleotidet (96), hvori codon for Met er erstattet af Leu. .Example 12 CSF (1-35) Ile (37-45) Leu (47-78) Leu-Leu (81-127) The oligonucleotide (95) containing codon for Ile instead of Met is first synthesized. and the oligonucleotide (96), in which the codon for Met is replaced by Leu. .
Derpå åbnes plasmidet (72) (fig. 10) med Pvu I 35 og Xma III, og fragmentet (97) isoleres. Endvidere syntetiserer man sekvensen (98), i hvilket der forud for codon for ' 19 DK 170346 B1 0 den første aminosyre er anbragt codon for Met.Then, plasmid (72) (Fig. 10) is opened with Pvu I 35 and Xma III, and the fragment (97) is isolated. Further, the sequence (98) is synthesized in which prior codon for the first amino acid is codon for Met.
Ligerer man derpå fragmenterne (16), (98), (97), (95) og (96) , får man således plasmidet pS 208 (99). Dette svarer til plasmidet (72), men indeholder i CSF-I-sekven-5 sen i positionen 0 codon for Met, i position 36 et codon for Ile og i position 46 et codon for Leu.If fragments (16), (98), (97), (95) and (96) are then ligated, the plasmid pS 208 (99) is thus obtained. This is similar to the plasmid (72) but contains in the CSF-I sequence at position 0 codon for Met, at position 36 a codon for Ile and at position 46 a codon for Leu.
Man syntetiserer endvidere sekvensen (100), som i positionerne 79 og 80 koder for Leu i stedet for Met.Furthermore, the sequence (100), which in positions 79 and 80 encodes Leu instead of Met, is synthesized.
10 Åbner man plasmidet (76) (fig. 10) med Hind III10 Opens the plasmid (76) (Fig. 10) with Hind III
og Nhe I, isolerer det store fragment (101) og ligerer det med den syntetiske sekvens (100), får man således plasmidet pS 209 (102), som svarer til plasmidet (76) på nær udbytningen af de to codoner i position 79 og 80 i CSF-III-15 -sekvensen.and Nhe I, isolating the large fragment (101) and ligating it to the synthetic sequence (100) thus obtains the plasmid pS 209 (102) corresponding to the plasmid (76) except for the exchange of the two codons at position 79 and 80 in the CSF-III-15 sequence.
Man spalter derpå plasmidet (93) (fig. 11a) partielt med Pvu I og med Sal I og isolerer det store fragment (103). Plasmidet (99) åbnes ligeledes partielt med Pvu I og med Pst I, og man isolerer det lille fragment (104) 20 som indeholder den modificerede CSF-I-sekvens. Desuden åbner man plasmidet (102) med Hind III og Sal I og isolerer det lille fragment (105) , som omfatter den modificerede CSF-III-sekvens.The plasmid (93) (Fig. 11a) is then partially cleaved with Pvu I and Sal I and the large fragment (103) is isolated. Plasmid (99) is also partially opened with Pvu I and with Pst I, isolating the small fragment (104) 20 containing the modified CSF-I sequence. In addition, the plasmid (102) is opened with Hind III and Sal I and isolated the small fragment (105) comprising the modified CSF-III sequence.
Derpå ligerer man fragmenterne (103), (104), Ϊ70) 25 og (105), hvorved man får plasmidet pS 210 (106), som svarer til plasmidet (93) (fig. 11a), men koder for et CSF-derivat, i hvilket Met står i positionen 0, og på den anden side er de fire Met-grupper erstattet af andre aminosyrer.Then, the fragments (103), (104), Ϊ70) 25 and (105) are ligated to give the plasmid pS 210 (106), which corresponds to the plasmid (93) (Fig. 11a), but encodes a CSF derivative , in which Met is in position 0, and on the other hand, the four Met groups are replaced by other amino acids.
Transformerer man med plasmidet (106) E. coli, 30 får man således efter induktion et fusionsprotein, som er spalteligt med cyanohalogenid, idet man får et CSF-derivat, som i position 36 indeholder Ile og i positionerne 46, 79 og • 80 Leu.Thus, when transformed with the plasmid (106) E. coli, 30, upon induction, a fusion protein cleavable with cyanohalide is obtained, yielding a CSF derivative containing at position 36 Ile and at positions 46, 79 and 80 Leu. .
35 DK 170346 B1 20 o35 DK 170346 B1 20 o
Eksempel 13 CSF(1-35)Ile(37-45)Leu(47-78)Leu(81-127) Går man frem ifølge eksempel 12, men i stedet for den syntetiske sekvens (100) anvender den syntetiske sekvens 5 (107), får man således et deletionsprodukt, hvori der i po sition 36 står Ile og i position 46 Leu, og hvori der i stedet for aminosyrerne79 og 80 forekommer aminosyren Leu.Example 13 CSF (1-35) Ile (37-45) Leu (47-78) Leu (81-127) Proceed according to Example 12, but instead of the synthetic sequence (100), the synthetic sequence 5 (107) is used. ), there is thus obtained a deletion product in which at position 36 stands Ile and at position 46 Leu and in which amino acids Leu are substituted for amino acids79 and 80.
10 Eksempel 14 CSF(1-35)Ile(37-45)Leu(47-78)-(81-127) Går man frem ifølge eksempel 12, men i stedet for den syntetiske sekvens (100) indsætter den syntetiske sekvens (108) får man således et deletionsprodukt, hvori der 15 i position 36 står Ile og i position 46 Leu, og aminosyrerne i positon 79 og 80 er fjernede.Example 14 CSF (1-35) Ile (37-45) Leu (47-78) - (81-127) Proceed according to Example 12, but instead of the synthetic sequence (100) insert the synthetic sequence (108 ) thus obtains a deletion product wherein 15 at position 36 stands for Ile and at position 46 Leu, and the amino acids at positions 79 and 80 are removed.
20 25 " 30 35 21 DK 170346 B1 020 25 "30 35 21 DK 170346 B1 0
BilagAppendix
Synteseblok I (CSF-I) (69) 5 --Ici-----Ic 1 · ·Synthesis Block I (CSF-I) (69) 5 - Ici ----- Ic 1 · ·
• AAT TCG ATC GAC GAC CCG GCG CCG GCcJcGA TCG CCG TCT CCG• AAT TCG ATC GAC GAC CCG GCG CCG GCcJcGA TCG CCG TCT CCG
GC TAG CTG CTG GGC CGC GGC CGG GCT AGcJgGC AGA GGCGC TAG CTG CTG GGC CGC GGC CGG GCT AGcJgGC AGA GGC
(Eco Ri) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro(Eco Ri) Ile Asp Asp Pro Ala Pro Ala Arg Ser Pro Ser Pro
io »--xt—LU_122. ^-1Jio »--xt — LU_122. ^ -1J
t 50 XC — ► - 2e - -—X-3t 50 XC - ► - 2nd - -—X-3
TCG ACC CAG CCC |tGG GAA CAC GTT AAC GCG AT C CAG GaJa GCG 15 AGC TGG GTC GGG ACC CTT|GTG CAA TTG CGC TAG GTC CTT CGCTCG ACC CAG CCC | tGG GAA CAC GTT AAC GCG AT C CAG GaJa GCG 15 AGC TGG GTC GGG ACC CTT | GTG CAA TTG CGC TAG GTC CTT CGC
Ser Thr Gin Pro Trp Glu His Val Asn Ala Ile Gin Glu Ala τά—^ι--m_ifSer Thr Gin Pro Trp Glu His Val Asn Ala Ile Gin Glu Ala τά— ^ ι - m_if
1\--—-—-H1 \ H ------
20 c · · · ·20 c · · · ·
CGG CGTCTG CTG AAC CTG AGT CGC|GAC ACG GCC GCG GAA ATGCGG CGTCTG CTG AAC CTG AGT CGC | GAC ACG GCC GCG GAA ATG
Gcjc GCA GAC GAC TTG GAC TCA GCG CTG TGC | CGG CGC CTT TACGcjc GCA GAC GAC TTG GAC TCA GCG CTG TGC | CGG CGC CTT TAC
Arg Arg Leu leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met li—«-£22)-Xf,-(30)-^ ^-(35)-1 k 25 --U_!« • · · · ·Arg Arg Leu leu Asn Leu Ser Arg Asp Thr Ala Ala Glu Met li - «- £ 22) -Xf, - (30) - ^ - - (35) -1 k 25 --U_!« • · · · ·
AAC GAA ACC GTT GAAJgTGATA TCT GAG ATG TTC GAC CTG CAAAC GAA ACC GTT GAAJgTGATA TCT GAG ATG TTC GAC CTG CA
TTG CTT TGG CAA CTT CAC TATjAGA CTC TAC AAG CTG G (Pst I) 30 Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe Asp(leu) T/ (40) (45) (50)TTG CTT TGG CAA CTT CAC TATjAGA CTC TAC AAG CTG G (Pst I) 30 Asn Glu Thr Val Glu Val Ile Ser Glu With Phe Asp (leu) T / (40) (45) (50)
Xk----► ----p* 35 * 22 DK 170346 B1 0Xk ---- ► ---- p * 35 * 22 DK 170346 B1 0
Synteseblok II (CSF-II) (70) 150 ^ τη- .Synthesis block II (CSF-II) (70) 150 ^ τη-.
--Ha- ---JlC- HA- --- JlC
5 · · · · ** (Pst i)g gaa ccg aca tgt ctc cag acg|cgt ctc gag ctc tac5 · · · · ** (Pst i) g gaa ccg aca tgt ctc cag acg | cgt ctc gag ctc tac
AC GTC CTT GGC TGT ACA GAG GTC TGC GCA GAG|CTC GAG ATGAC GTC CTT GGC TGT ACA GAG GTC TGC GCA GAG | CTC GAG ATG
(Gln)Glu Pro Thr Cys Leu Gin Thr Arg Leu Glu leu Tyr (50) __ (55) (60) _ .(Gln) Glu Pro Thr Cys Leu Gin Thr Arg Leu Glu leu Tyr (50) __ (55) (60) _.
10 JLL ia 1 " pm ............... II Cl _ 200 w 214 n c--- -*-jPe-►*> AAA CAA GGCjCTT CGT GGT TCT CTG ACC A (Hind III)10 JLL ia 1 "pm ............... II Cl _ 200 w 214 n c --- - * - jPe-► *> AAA CAA GGCjCTT CGT GGT TCT CTG ACC A ( Hind III)
TTT GTT CCG GAA GCAlCCA AGA GAC TGG TTC GATTT GTT CCG GAA GCAlCCA AGA GAC TGG TTC GA
15 lys Gin Gly Leu Arg Gly Ser Leu Thr (Lys)15 Lys Gin Gly Leu Arg Gly Ser Leu Thr (Lys)
Icl-<«>---iZSljrf-- 20 25 30 > 35 0 23 DK 170346 B1Icl - <«> --- iZSljrf-- 20 25 30> 35 0 23 DK 170346 B1
Synteseblok III (CSF-III) (71) 215 ΤΓ 250 _ ^—......ilia-----JITc • · · · ·Synthesis Block III (CSF-III) (71) 215 ΤΓ 250 _ ^ —...... ilia ----- JITc • · · · ·
5 , AG CTT AAG GGG CCC CTC ACC ATG ATG GCT AGC CAc|tAC AAA5, AG CTT AAG GGG CCC CTC ACC ATG ATG GCT AGC CAc | tAC AAA
("Hinå III) A TTC CCC GGG GAG TGG TAC TAC CGA TCG GTG ATG TTT | (Leu)lys Gly Pro leu Thr Met Met Ala Ser His Tyr lys (72) (75) (80) (85) --BTfc--- 10 ffic - - - ***· --UTe("Hinå III) A TTC CCC GGG GAG TGG TAC TAC CGA TCG GTG ATG TTT | (Leu) list Gly Pro leu Thr Met With Ala Ser His Tyr list (72) (75) (80) (85) --BTfc- - 10 ffic - - - *** · --UTe
CAG CAC TGC CCG CCG ACT CCG GAG ACg|tCT TGC GCA ACG CAG GTC GTG ACG GGC GGC TGA GGC CTC TGC AGA ACg]CGT TGC GTCCAG CAC TGC CCG CCG ACT CCG GAG ACg | tCT TGC GCA ACG CAG GTC GTG ACG GGC GGC TGA GGC CTC TGC AGA ACg] CGT TGC GTC
Gin His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gin ,5 ---^£L_sN-r(95) ► *-sr-f 300 _ jTe-7—-;-J-ϋί-3-;--Gin His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr Gin, 5 --- ^ £ L_sN-r (95) ► * -sr-f 300 _ jTe-7 —-; - J-ϋί-3 -; - -
ATO ATCIACC TTO GAA TOT TTT AAA GAA AAC CTS AAG GAC ITTATO ATCIACC TTO GAA TO TTT AAA GAA AAC CTS AAG GAC ITT
TAG TAG TGG AA&I CTT AGA AAA TTT CTT TTG GAC TTC CTG AAATAG TAG TGG AA&I CTT AGA AAA TTT CTT TTG GAC TTC CTG AAA
2020
Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe _-Ooo) (105) (110) j!Lf--Hi 350 391 --Ml i------nrk • · · · ·· ^Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys Asp Phe _-Ooo) (105) (110) j! Lf - Hi 350 391 --Ml i ------ nrk • · · · ·· ^
25 |cTG CTT GTT ATA CCG TTC GAC TGT TGG GAG|CCG GTC CAG GAA25 | cTG CTT GTT ATA CCG TTC GAC TGT TGG GAG | CCG GTC CAG GAA
GAC GAA|CAA TAT GGC AAG CTG ACA ACC CTC GGC CAG|GTC CTTGAC GAA | CAA TAT GGC AAG CTG ACA ACC CTC GGC CAG | GTC CTT
Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gin Glu (115) (120) (125) .Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gin Glu (115) (120) (125).
Dpi--l&j -► --/II iDpi - l & j -► - / II i
30 V30 V
Mk— »Mk— »
Sal I Pst I TGA TA*G TCG ACT GCA GCC C ACT ATC AGC TGA CGT CGG G 35 Stp Stp (Sma I)Will I Pst I TGA TA * G TCG ACT GCA GCC C ACT ATC AGC TGA CGT CGG G 35 Stp Stp (Sma I)
Ri ^Ri ^
Claims (8)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3545568 | 1985-12-21 | ||
DE19853545568 DE3545568A1 (en) | 1985-12-21 | 1985-12-21 | GM-CSF-PROTEIN, ITS DERIVATIVES, PRODUCTION OF SUCH PROTEINS AND THEIR USE |
Publications (3)
Publication Number | Publication Date |
---|---|
DK619086D0 DK619086D0 (en) | 1986-12-19 |
DK619086A DK619086A (en) | 1987-06-22 |
DK170346B1 true DK170346B1 (en) | 1995-08-07 |
Family
ID=6289242
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
DK619086A DK170346B1 (en) | 1985-12-21 | 1986-12-19 | Granulocyte Macrophage Colony Stimulating Factor (CSF) Proteins, Method for Preparation thereof, Expression Vector and E.coli Cell for Use in This Method and Using These Proteins |
Country Status (17)
Country | Link |
---|---|
EP (1) | EP0228018B1 (en) |
JP (1) | JP2575367B2 (en) |
KR (1) | KR940005585B1 (en) |
AT (1) | ATE71144T1 (en) |
AU (2) | AU601959B2 (en) |
CA (1) | CA1341197C (en) |
DE (2) | DE3545568A1 (en) |
DK (1) | DK170346B1 (en) |
ES (1) | ES2055686T3 (en) |
FI (1) | FI91168C (en) |
GR (1) | GR3003999T3 (en) |
HU (1) | HU202584B (en) |
IE (1) | IE59779B1 (en) |
IL (1) | IL81020A (en) |
NO (1) | NO177270C (en) |
PT (1) | PT83972B (en) |
ZA (1) | ZA869557B (en) |
Families Citing this family (20)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5078996A (en) * | 1985-08-16 | 1992-01-07 | Immunex Corporation | Activation of macrophage tumoricidal activity by granulocyte-macrophage colony stimulating factor |
AU606585B2 (en) * | 1985-10-03 | 1991-02-14 | Biogen, Inc. | Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells |
DE3712985A1 (en) * | 1987-04-16 | 1988-11-03 | Hoechst Ag | BIFUNCTIONAL PROTEINS |
US5359035A (en) * | 1985-12-21 | 1994-10-25 | Hoechst Aktiengesellschaft | Bifunctional proteins including interleukin-2 (IL-2) and granuloctyte macrophage colony stimulating factor (GM-CSF) |
JP2583770B2 (en) * | 1986-09-17 | 1997-02-19 | 大塚製薬株式会社 | gene |
JPS6420097A (en) * | 1987-03-02 | 1989-01-24 | Sumitomo Chemical Co | Human granulocyte-macrophage colony stimulating factor |
PT87133B (en) * | 1987-04-02 | 1992-07-31 | Amrad Corp Ltd | METHOD OF PURIFICATION OF THE LEUKEMIA INHIBITOR FACTOR (LIF) AND PHARMACEUTICAL COMPOSITIONS CONTAINING POLIPEPTIDES WITH LIF ACTIVITY |
AU609128B2 (en) * | 1987-04-02 | 1991-04-26 | Amrad Operations Pty. Limited | Leukaemia-inhibitory factor |
EP0383764B1 (en) * | 1987-06-25 | 1995-04-12 | Immunex Corporation | Bovine granulocyte-macrophage colony stimulating factor |
AU638204B2 (en) * | 1987-11-12 | 1993-06-24 | Schering Corporation | Acceleration of bone formation with gm-csf |
GB2212159B (en) * | 1987-11-13 | 1992-01-22 | British Bio Technology | Synthetic gene for human granulocyte/macrophage colony stimulating factor. |
WO1989010403A1 (en) * | 1988-04-21 | 1989-11-02 | Medvet Science Pty. Ltd. | Human gm-csf variants |
AU5355790A (en) * | 1989-04-19 | 1990-11-16 | Cetus Corporation | Multifunctional m-csf proteins and genes encoding therefor |
DE69007975T2 (en) * | 1989-08-22 | 1994-07-21 | Immunex Corp | FUSION PROTEIN CONSISTING OF GM-CSF AND IL-3. |
NZ236819A (en) * | 1990-02-03 | 1993-07-27 | Max Planck Gesellschaft | Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions |
US5270181A (en) * | 1991-02-06 | 1993-12-14 | Genetics Institute, Inc. | Peptide and protein fusions to thioredoxin and thioredoxin-like molecules |
EP0872557A1 (en) * | 1995-09-28 | 1998-10-21 | Otsuka Pharmaceutical Co., Ltd. | Neutrophil chemotactic lymphokine, and drug and kit for the diagnosis of drug hypersensitive granulocytopenia containing the same |
KR100448021B1 (en) * | 2001-12-28 | 2004-09-08 | 크레아젠 주식회사 | Transgenic Escherichia coli transformed with pGM-CSF vector expressing recombinant mouse granulocyte-macrophage colony stimulating factor and method for mass production of the recombinant protein |
CN108295087B (en) * | 2009-12-23 | 2021-01-15 | 格兰达利斯有限公司 | Furin-knockdown and GM-CSF-enhanced (FANG) cancer vaccines |
SG181881A1 (en) | 2009-12-23 | 2012-07-30 | Gradalis Inc | Furin-knockdown bi-functional rna |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4438032A (en) * | 1981-01-30 | 1984-03-20 | The Regents Of The University Of California | Unique T-lymphocyte line and products derived therefrom |
SE8300693L (en) * | 1983-02-09 | 1984-08-10 | Sven Lofdahl | SET TO MAKE AND ISOLATE PROTEINS AND POLYPEPTIDES, AND A HYBRID VECTOR FOR THIS |
IL71991A (en) * | 1983-06-06 | 1994-05-30 | Genentech Inc | Preparation of mature human IGF and EGF via prokaryotic recombinant DNA technology |
AU594014B2 (en) * | 1984-03-21 | 1990-03-01 | Research Corporation Technologies, Inc. | Recombinant DNA molecules |
DE3588199T2 (en) * | 1984-07-06 | 1999-04-08 | Novartis Ag, Basel | Lymphokine production and purification |
ZA856108B (en) * | 1984-10-29 | 1986-10-29 | Immunex Corp | Cloning of human granulocyte-macrophage colony simulating factor gene |
AU588819B2 (en) * | 1984-10-29 | 1989-09-28 | Immunex Corporation | Cloning of human granulocyte-macrophage colony stimulating factor gene |
DE3586826T2 (en) * | 1984-11-20 | 1993-03-25 | Schering Biotech Corp | CDNA CLONES ENCODING PEPTIDES WITH THE EFFECTIVENESS OF A HUMAN GRANULOCYTE, MACROPHAGE AND EOSINOPHILE CELL GROWTH FACTOR. |
AU606585B2 (en) * | 1985-10-03 | 1991-02-14 | Biogen, Inc. | Human granulocyte-macrophage colony stimulating factor-like polypeptides and processes for producing them in high yields in microbial cells |
DE3541856A1 (en) * | 1985-11-27 | 1987-06-04 | Hoechst Ag | EUKARYOTIC FUSION PROTEINS, THEIR PRODUCTION AND USE, AND MEANS FOR CARRYING OUT THE PROCESS |
DE3636903A1 (en) * | 1985-12-21 | 1987-07-02 | Hoechst Ag | FUSION PROTEINS WITH EUKARYOTIC BALLASTES |
-
1985
- 1985-12-21 DE DE19853545568 patent/DE3545568A1/en not_active Withdrawn
-
1986
- 1986-12-16 EP EP86117484A patent/EP0228018B1/en not_active Expired - Lifetime
- 1986-12-16 AT AT86117484T patent/ATE71144T1/en not_active IP Right Cessation
- 1986-12-16 ES ES86117484T patent/ES2055686T3/en not_active Expired - Lifetime
- 1986-12-16 DE DE8686117484T patent/DE3683267D1/en not_active Expired - Lifetime
- 1986-12-18 FI FI865186A patent/FI91168C/en not_active IP Right Cessation
- 1986-12-18 IL IL8102086A patent/IL81020A/en active IP Right Grant
- 1986-12-19 NO NO865191A patent/NO177270C/en unknown
- 1986-12-19 HU HU865355A patent/HU202584B/en not_active IP Right Cessation
- 1986-12-19 DK DK619086A patent/DK170346B1/en not_active IP Right Cessation
- 1986-12-19 CA CA000525856A patent/CA1341197C/en not_active Expired - Fee Related
- 1986-12-19 IE IE333586A patent/IE59779B1/en not_active IP Right Cessation
- 1986-12-19 AU AU66756/86A patent/AU601959B2/en not_active Ceased
- 1986-12-19 PT PT83972A patent/PT83972B/en not_active IP Right Cessation
- 1986-12-19 ZA ZA869557A patent/ZA869557B/en unknown
- 1986-12-20 KR KR1019860011011A patent/KR940005585B1/en not_active IP Right Cessation
- 1986-12-22 JP JP61306186A patent/JP2575367B2/en not_active Expired - Lifetime
-
1990
- 1990-09-19 AU AU62683/90A patent/AU637139B2/en not_active Ceased
-
1992
- 1992-03-11 GR GR920400222T patent/GR3003999T3/el unknown
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Legal Events
Date | Code | Title | Description |
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B1 | Patent granted (law 1993) | ||
PBP | Patent lapsed |