WO1989002462A1 - Plasmides recombinants produisant l'hormone d'adenocorticotropique humaine (acth) - Google Patents

Plasmides recombinants produisant l'hormone d'adenocorticotropique humaine (acth) Download PDF

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WO1989002462A1
WO1989002462A1 PCT/DE1988/000536 DE8800536W WO8902462A1 WO 1989002462 A1 WO1989002462 A1 WO 1989002462A1 DE 8800536 W DE8800536 W DE 8800536W WO 8902462 A1 WO8902462 A1 WO 8902462A1
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acth
psa
coli
plasmid
dna
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Werner Boidol
Joachim Daum
Peter Donner
Hartmut Seliger
Gerhard Siewert
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Schering Aktiengesellschaft
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/695—Corticotropin [ACTH]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • Re orabinante plasmids for the production of human adrenocorticotropic hormone (ACTH).
  • ACTH adrenocorticotropic hormone
  • corticotrophin corticotrophin
  • ACTH regulates the secretion of glucocorticoids, in particular hydrocortisone and cortisone, in the zona fasciculata of the adrenal cortex and thus influences the carbohydrate, protein and fat metabolism in a complex manner.
  • ACTH has a regulating effect on the production of sex hormones in the zona reticularis, which directly surrounds the adrenal center as the inner layer of the bark.
  • ACTH In human medicine, ACTH is used therapeutically to treat inflammatory and allergic processes such as rheumatic fever or bronchial asthma. It is also important in the clinic for the diagnosis of adrenal cortex function.
  • inflammatory and allergic processes such as rheumatic fever or bronchial asthma. It is also important in the clinic for the diagnosis of adrenal cortex function.
  • preparations There are currently two types of preparations available:
  • ACTH from animal sources especially enriched extracts from porcine pituitary glands.
  • Human ACTH differs from porcine ACTH only in one amino acid (see Teh H. Lee et al., I.e.). The latter contains a leucyl residue in position 31 instead of the seryl residue in the human sequence.
  • genetic engineering has the primary task of developing the range of processes for in-vitro processing of genetic material, i.e. to develop and expand DNA fragments with very different physico-chemical and biological properties.
  • a DNA fragment which contains the desired genetic information. There are several possibilities for that. In the simplest case, it can be Cut out the table from the DNA of a suitable donor species, a frequently used method if the donor is a prokaryote. On the other hand, a gene can be synthesized enzymatically by reverse transcription from messenger RNA (cDNA method). Another possibility is the chemical synthesis of oligonucleotides and their enzymatic linkage to the desired gene fragment.
  • the desired DNA fragment is enzymatically integrated into a so-called vector DNA (for details, see the literature references mentioned above).
  • the resulting gene construction has the necessary functions for introducing the foreign gene into suitable host cells and for its stable establishment in an autonomously replicating expression unit or built into the host chromosome, as well as marker functions for the selection of successfully transformed in vitro recombined clones.
  • Prokaryotic host organisms are particularly attractive for the development of biotechnological processes with genetically engineered expression clones. They can be fermented relatively inexpensively using established process technologies and in some cases have been genetically well studied. The latter applies especially to the intestinal bacteria Escherichi coli and derived apathogenic laboratory strains such as E.col K12.
  • fusion with host-specific genes and the resulting expression as fusion proteins permits the biotechnological synthesis of short-chain target peptides in E. coli [for example, J. Shine et al., Nature 2Z5_ r 456-461 (1980)].
  • Such small peptides cannot be directly synthesized in E. coli cells because they do not have a fixed spatial structure and are therefore preferentially degraded by host proteases present in cytoplasm [cf. K. Itakura et al., Science 198, 1056-1063 (1977)].
  • parts of fusion proteins they can be obtained in good yields under suitable conditions.
  • Fusion proteins that are to be specifically split by this method must be constructed in such a way that between the amino end of the target peptide and the carboxyl end of the bacterial portion there is an at least repeated tetrapeptide recognition sequence for collagenases of the clo-type. stridiopeptidase A is located.
  • Such constructions have the general structure:
  • n 1 applies, but for most examples n> 1.
  • the target cleavage product obtained is the target peptide extended by the dipeptide Gly-Pro at the N-terminal, which can be quantitatively converted into the native form by cleaving off the glycylproline by means of postproline dipeptidyl aminopeptidase (PPDA) (see FIG EP 20290).
  • PPDA postproline dipeptidyl aminopeptidase
  • the existing recognition sequence for collagenase offers the possibility for the enzymatic release of Gly- Pro-ACTH, Gly-Pro-ACTH 1-24 or other ACTH derivatives, as already mentioned above, which can be split further with PPDA to the native proteohormones (cf. EP 20290).
  • Fusion proteins such as the ACTH clones described in step 1. In contrast to this, however, they permit a decisive release for biotechnology, which is accelerated by orders of magnitude (cf. Tab. 2) and quantitative release of z.
  • the present invention relates to plasmid vectors which contain a DNA sequence for ACTH or ACTH derivatives and with which bacteria can be transformed into cells which contain these vectors.
  • the invention further relates to methods for producing these plasmid vectors, which are characterized in that DNA insertion fragments, which contain the codons for ACTH or ACTH derivatives and which have Pstl recognition sequences at both ends, are linearized into the Pstl interface and dephosphorylated Inserts vector plasmids with the aid of suitable ligases and E. coli strains containing plasmids with a Ge for ACTH or ACTH derivatives.
  • the present invention relates to processes for the production of new E.
  • the invention also encompasses a process for the production of new E. coli strains by processes known per se, which is characterized in that competent cells from E. coli E 15 are extracted with a high excess of plasmid DNA for ACTH or ACTH derivatives E. coli SB transformed, and the use of these transformed E. coli E 15 strains for the production of ACTH and ACTH derivatives.
  • processes for the production of ACTH may be mentioned which are characterized in that the new strains (see claims 13-26) are obtained from a preculture with an aqueous solution of L-proline, L-leucine, casatino acids, vitamin B 1 in concentrated low phosphate medium combined in a fermenter, stirred at 37 ° C.
  • the invention relates to medicaments containing ACTH or ACTH derivatives, which can be prepared by the processes described above, and customary auxiliaries and carriers.
  • Escherichia coli SB 44 (pSA 125) (DSM 4191) Escherichia coli HB 101 (pSA 504) (DSM 4192) Escherichia coli HB 101 (pSA 271) (DSM 4193)
  • Plasmid pBR 322 (ATCC 40015)
  • Escherichia coli SB 44 (pSB 53) (ATCC 31542)
  • alkaline phosphatase from calf intestine Boehringer / Mannheim
  • T4 polynucleotide kinase Pharmacia / PL, 7800 Freiburg i. Br. Host strains for in vitro recombined DNA constructions:
  • the resulting radioactive DNA fragment was separated by means of gel electrophoresis in 0.1 mm thin, thermostated (60 ° C.) polyacrylamide / urea gels.
  • To sorge and L. de Maeyer J. Chromate 202, 45-53, (1980)] using the Macrophor separation system from LKB Instructions.
  • Goat anti-rabbit IgG conjugated with alkaline phosphatase was used as the second antibody [Fa. Cappel represented by Cooper Biomedical, 6000 Frankfur / Main 1]
  • the color reaction was carried out by adding 5-bromo-4-chloro-3 indolyl phosphate.
  • the insoluble cell disruption products are sedated by means of centrifugation at 0 ° C. and 48,000 ⁇ g for 20 minutes. By resuspending twice in 20 ml of 50 mM Tris / HCl pH 8 160 mM NaCl and 10 min. Recentrifugation under the above conditions, the sediments can be obtained free of soluble cellulose substances. They are resuspended in 3 ml of 8 M guanidine * HC1 using a dounce homogenizer, heated to 95 ° C. for 1 hour and then cooled to ice temperature. ACTH fusion proteins dissolve stably under these conditions and can be obtained as supernatant after 30 min centrifugation under the above conditions.
  • the proteins After 16 hours of dialysis at 2 ° C. against 4 1 50 mM Tris HC1 pH 8, 160 mM NaCl, the proteins are obtained in 4 ml dia lysate as a finely dispersed suspension, which in this form can be used directly for collagenase digestion or can be stored at -20 ° C.
  • the specific cleavage of the ACTH fusion proteins is expediently carried out using a highly purified collagenase preparation which can be determined by FPLC ion exchange chromatography [cf. Deu patent application file number P 37 31 875.6] of the commercially available Clostridiopeptidase A (EC 3.4.24.3) from Clostri dium histolyticum [Type VII; Sigma GmbH].
  • i main culture that is 4 ⁇ l protein suspension (dialysate), in 10 ⁇ l 50 M Tris / HCl pH 8, 5 mM CaCl- with desired units [cf. E. Wünsch et al., Hoppe Seyler's Z Physiol. Chem. 333, 149-151 (1963)] Clostridiopeptidase A incubated for 1 h at 37 ° C.
  • High buffer 100 mM NaCl, 50 mM Tris / HCl, -10 mM mgCl 2 ,
  • High buffer 100 mM NaCl, 50 mM Tris / HCl, 10 mM gCl 2 ,
  • This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-446 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by the collagenase recognition sequence -Pro-Ala-Gly-Pro and the ACTH-24 sequence (SA 172):
  • the DNA was dissolved in 5 ⁇ l TE buffer (solution B 2.) and 10 ⁇ l reaction volume with alkaline phosphatase from the small intestine according to Maniatis et al. (Solutions according to B 1.2.) Depho-phorylated. After extraction with 300 ⁇ l TE-saturated pheno and twice extraction with 600 ⁇ l ether each, 150 were made of NaCl and precipitated with 0.5 ml ethanol (as described in C 1.1.1. B) and sedimented.
  • 0.5 ⁇ g of desphosphorylated pSA 125 vector DNA were m 0.5 ⁇ g of the isolated 86 bp fragment in 100 ⁇ l ligase powder (solution B 1.3.) With 4.5 Weiss units [B. Weiss et al. J. Biol. Chem. 243, 4543-4555 (1968)] T4 DNA ligase incubated for hours at 23 ° C. The reaction mixture was used for the transformation of competent recipient cells.
  • the ampicillin resistance is retained when it is incorporated into the desired Pstl site in positi 1624 of the vector.
  • the transformation mixture was therefore placed on LB agar [T. M niatis, E.F. Fritsch and J. Sambrook in: 'Molecular Cloning' p. 440, Cold Spring Harbor Lab., New York (1982)], which contained 100 ⁇ g / ml ampicillin. Twelve clones were selected from 220 transformant colonies for the isolation of plasma mid-DNA.
  • REPLACEMENT LEAF C 1.3 Characterization of transformants by size determination of DNA fragments after implementation with special restriction endonucleases
  • the plasmid isolation from 12 randomly selected clones was carried out according to standard conditions [cf. A 1.]. By digesting back with PstI, the incorporation of the 86 bp fragment could be ensured in all cases.
  • This clone synthesizes a fusion protein consisting of d N-terminal amino acids 1-446 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by the Collagenaseseque -Pro-Ala-Gly-Pro and the ACTH sequence (SA 186) consists of:
  • a 131 bp DNA fragment with PstI recognition sequences at the ends, which contains the codons for ACTH [cf. Fig. 4a ACTH sequence] was from plasmid pSA 504 [deposited as E. coli HB 101 (pSA 504) - DSM 4192].
  • Clones which contain the ACTH insertion fragment inserted in the desired phoA transcription direction have fragments of
  • This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by glycine and 5 collagenase identification sequences before the ACTH sequence (SA 341):
  • REPLACEMENT LEAF C 3.1 Single or multiple integration of 131 bp-Pstl restriction fragments with codons for ACTH into the Pstl interface in position 1657 of the vector plas mids pSA 506
  • the entire ligase reaction mixture was used to transform competent E. coli SB 44 cells as described under 1.2.1. refereed, used.
  • this oligonucleotide is complementary over a region of 41 bases (90%) to the gene segment in the information strand of the plas ids pSA 341 [Fig. 7], which for the 5 consecutive collagenase recognition sequences in the fusion protein [cf. C 3.] coded.
  • This clone synthesizes a fusion protein which consists of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by glycine and 2 collagenase recognition sequences before the ACTH sequence (SA 343), consists of:
  • This clone synthesizes a fusion protein consisting of d amino acids 1-444 of the prepeptide of alkaline phosphates [M. Simonis, dissertation Freie (2015) Berlin, Berl (1983)], followed by glycine and 5 collagenase recognition sequences before the ACTH 1-24 sequence (SA 353), consists of:
  • This clone synthesizes a fusion protein which consists of d N-terminal amino acids 1-444 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1982)], followed by glycine and 5 collagenase-E
  • the DNA was dissolved in 26 ⁇ l TE buffer (solution B 2.) and in 30 ⁇ l reaction volume, as in C 1.1.2. described, dephosphorylated and processed.
  • This fragment could be ligase-linked to a sub-fragment from pSA 186 [cf. Fig. 5] with 2 synthetically manufactured oligonucleotides.
  • the two oligonucleotides are complementary to one another and, under suitable conditions, form a 45 bp-Pstl / Ncol hybrid. Synthesized and worked up under standard conditions [cf. A 3.] became the 41 base nucleotide
  • the ligase products were incubated in 100 ⁇ l of medium buffer (solution 1.1.) With 9 units of PstI [Boehringer / Mannheim] for 3 hours at 37 ° C. and again according to C 7.1.3.3. worked up
  • the plasmid isolation from 16 clones selected at random was carried out under standard conditions [cf. A I.].
  • test clone (pSA 509) showed the typical Ncol band pattern for correct installation.
  • This clone synthesizes a ⁇ fusion protein which consists of the N-terminal amino acids 1-297 of the prepeptide of the alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by proline, tryptophan and 4 collagenase recognition sequences before the ACTH sequences (HS 6134), consists of:
  • 50 ⁇ g pSA 509-DNA are made according to C 1.1.3. digested with PstI and worked up after gel electrophoretic isolation.
  • dephosphorylated pSA 302 vector DNA was 0.25 ⁇ g of the isolated 158 bp fragment in 50 ⁇ l Ligase-P fer (solution B 1.3.) with 1 white unit [B. Weiss et al., J. Biol. Chem. 243, 4543-4555 (1968)] T4 DNA ligase [Boehringer / Mannheim] incubated overnight at 16 ° C.
  • the entire ligase reaction mixture from C 8.1.4. was acc. C 1.2.1. used to transform competent E.coli SB 44 cells.
  • the ampicillin resistance is retained when incorporated into the desired Pstl interface in position 1183 of the vector.
  • the transformer selection could therefore according to C 1.2.2. 15 ampicillin-resistant clones were selected for further investigations.
  • test clones showed the typical band pattern for correct installation. They received the designations pHS 6134 and pHS 613
  • This clone synthesizes a fusion protein which is identical to the product expressed by SB 44 (pHS 6134) [cf. C 8.1.
  • the expression plasmid pSA 351 contains, compared to the plasmid pHS 6134, a deletion of 461 base pairs in the non-translated DNA region between the ocher codon of the ACTH and the terminator region [cf. Chung Nan Chang et al. , Gene 44 121-125 (1986)] of the phoA gene.
  • the ligase products according to C 9.1.3. were completely transformed into competent cells from E. coli SB 44 under de by M. Dagert and S.D. Ehrlich [Gene 6_, 23-28 (1979)] described conditions used.
  • the DNA-treated cell suspension was divided into 5 aliquots to inoculate 20 ml of LB medium [T. Maniatis E.F. Fritsch and J. Sambrook in: 'Molecular Cloning', p. 440 Cold Spring Harbor Lab., New York (1982)] with 50 ⁇ g / ml Amp cillin. After 16 hours of shaking incubation at 37 °, diluted aliquots were plated on LB agar and after 24 hours of incubation at 37 ° C. 6 plates with 70-80 ampicin-resistant colonies each were selected for the characterization experiments.
  • the reaction was carried out analogously to C 9.3.2.1. carried out.
  • the enzyme activity used was 15 units Sau3AI [Boehringer / Mannheim].
  • This clone synthesizes a fusion protein consisting of the N-terminal amino acids 1-444 of the prepeptide of alkaline phosphatase [M. Simonis, dissertation Freie (2015) Berlin, Berlin (1983)], followed by glycine and 8 collagenase recognition sequences before the ACTH sequence (SA 360), consists of:
  • This plasmid differs from pSA 506 [cf. Fig. Only by a T / A—> C / G base exchange in Positi 1629, whereby the corresponding Pstl recognition sequence a pSA 506 is missing.
  • PSA 510 was produced in the same way as described for pSA 506 [see. German file number P 37 31 875.6].
  • pSB 94 produced according to P 37 31 875.6
  • two 5-base nucleotides with a correspondingly modified sequence were accordingly synthesized under standard conditions [cf. A 3]:
  • 0.7 ⁇ g of pSA 510 DNA were determined as in C 1.1.1. described, partially digested with 0.6 units of PstI [Boehringer / Mannheim] at 37 ° C. for 5 minutes and the reaction products, including the two 6499 bp-PstI fragments, according to C 1.1.1. worked up.
  • the required fragment was isolated from 50 ⁇ g pHS 6134 according to C 1.1.3 by Pstl digestion and subsequent processing.
  • test clones showed the typical Ncol band pattern for correct installation.
  • One of them was further cultivated under the name pSA 360 for the subsequent protein analysis.
  • E. c li E 15 has significant advantages over SB 44.
  • the TAG triplets used for the ACTH 1-24 hybrid genes pSA 172 and pSA 353 are, unlike in SB 44, strictly read as stop codons; on the other hand, the protein yields b E 15 clones are significantly higher under comparable culture conditions than with homologous SB 44 cloning.
  • 7 1 H_0 are expediently mixed with 2.2 1 of a sterile-filtered solution of 0.2 g L-proline, 0.2 g 'leucine, 10 g Casamino acids [Difco Lab. Detroit, USA] and 10 mg vitamin B 1 in 545-fold concentrated low-phosphate medium [cf. LP medium according to K. Kreuzer et al. , Genetics 8_1, 459-468 (1975)] and the 0.8 1 preculture combined in a closed glass fermenter with 15 1 capacity and for 6 hours at 37 ° C. with 10 1 air / min (0.7 atm pressure) and a stirrer speed stirred for 220 min.
  • the subsequent sedimentation of the bacterial cells by means of a "continuous centrifuge of the type Sharples T-1A [Deutsche Sharples GmbH, 4100 Duisburg-Meiderich] results in a cell yield of approx. 30 g wet weight.
  • the 30 g of low-phosphate cells are removed using an Ultra-Turrax T 25 [Fa. Jahnke & Kunke GmbH & Co., Kg, 7813 Staufen] in 300 ml 50 mM Tris / HC pH 7.5, 160 mM NaCl, 20 mM MgCl_, with 200 ⁇ DNase I [Boehringer / Mannheim], 300 ⁇ g pepstatin A un 10 mg phenyl-methyl-sulfonyl chloride (PMSF) [Sigma Chemie cf. A 6.3.] And temperature-controlled in the cold room to 2-4 ° C. A complete cell disruption under the mentioned temperature conditions can be achieved by using the high pressure homogenizer 15M-8TA [Fa. Manton Gaulin SA, Hilversum / NL] after 3 cycles at 9000-12000 psi. The temperature is then adjusted to DNase digestion at 0 ° C for 1 hour.
  • PMSF phenyl-methyl-sulfony
  • the sediment obtained is resuspended in 400 ml of 50 M Tris HC1 pH 8.0, 160 mM NaCl and sedimented as above. This washing process is repeated. Then the sediment free of soluble cell components is resuspended using Ultra-Turrax T 25 [Fa. Jahnke & Kunkel GmbH & Co. KG 7813 Staufen] in 100 ml 8 M guanidine hydrochloride in 50 m Tris / HCl pH 8.0 and heated to 95 ° C for 1 hour. Insoluble parts can be sedimented by a subsequent centrifugation of 20 minutes at 40,000 x g and are discarded
  • the clear 8 M guanidine chloride extract is first dialyzed against 2 1 H 0 for 30 min, then twice for 4 hours against 2 50 mM Tris / HCl pH8.0, 160 mM NaCl. This gives 70-80% pure fusion protein in insoluble form as finely dispersed suspension.
  • the dialysate suspension is mixed with 10 units of purified clostridiopeptidase A [see A 6.6.] And 15 ml of 0.5 M Tris / HCl pH 8.0, 50 mM CaCl 2 and with 50 mM Tris / HCl pH 8.0, 160 mM NaCl to 150 ml of reac volume added. This is followed by 3 hours of shaking incubation in a 37 ° C water bath. D 4.6. Processing of the reaction products
  • Insoluble residues of the collagenase digestion are sedimented by centrifugation at 45,000 x g for 30 minutes.
  • a 'Roto-Torque' mixer rotor [Fa. Cole Parmer Ins r. Co., Chicago, Illinois, USA] the clear centrifugation supernatant is mixed with 30 g of Mallinckrodt 100 esh silica gel [Serva Feinbiochemica GmbH & Co., 6900 Heidelberg 1 in a sealed bottle for 30 min. Rotation mixture [R.A. Donald, J. Endocrin 3_9_, 451-452 (1967)].
  • the adsorbent had previously been moistened by slurrying with H_0 and suction using a G3 glass frit.
  • the silica gel eluate is fixed on a PepRPC HR 16/10 column and with a linear 450 ml gradient of 10 mM KH-PO. in 0 (buffer A) - 50% (buffer B) acetonitrile 5 ml volumes at a flow rate of 5 ml / min. fractionally eluted.
  • the Gly-Pro-ACTH peak fractions can be identified photometrically at about 27.5% acetonitrile content (approx. 55% buffer B) after light absorption at 214 nm.
  • the reaction mixture is separated again using a PepRPC HR16 / 10 column under the conditions described in Section D 4.6.2. described technical conditions with the exception of the eluent.
  • the linear gradient consists of 225 ml 0.1% trifluoroacetic acid in H 2 0 (buffer A) and 225 ml 0.1% trifluoroacetic acid in 60% acetonitrile (buffer B).
  • the ACTH peak fractions can be identified photometrically in approximately 29.4% acetonitrile (approximately 49% buffer B) after light absorption at 214 nm (cf. Fig. 21).
  • Fig. 15 SDS gel electropherogram of protein extracts
  • E. coli SB 44 (phoA, supE) and E. coli E 15 (phoA) with the plasmids psB 94 [native phoA gene, cf. Parallel registration Dr. G. Siewert et al., (1987)], psA 172 (phoA / ACTH 1-24) and psA 186 (phoA / ACTH 1-39)
  • the clone expresses a fusion protein 27 amino acids long; MW 52 849
  • Fig. 17 Preparation of the pre-phoA / ACTH 1-39 fusion protein from
  • the amount of protein corresponds to 0.2 ml main culture.
  • Fig. 19 Clostridiopeptidase A digestion of the pre-phoA / ACTH 1-39 fusion proteins from E. coli E 15 with the plasmids pSA 341 and pSA 509, substrates and reaction products after SDS gel electrophoresis (12.5-25 7. polyacrylamide gradient)
  • Fig. 20 Clostridiopeptidase A digestion of the pre-phoA / ACTH 1-39 fusion proteins from E. coli E 15 with the plasmids pSA 341 and pSA 351 substrates and reaction products after SDS gel electrophoresis (12.5 7. polyacrylamide )

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Abstract

On produit l'ACTH et des dérivés d'ACTH biologiquement actifs par ingénierie génétique. On incorpore les séquences d'ADN correspondantes dans des plasmides vecteurs, qui à leur tour sont introduits dans les bactéries E.coli dans lesquelles ils sont exprimés. L'ACTH et ses dérivés ainsi obtenus sont utiles en tant que médicaments.
PCT/DE1988/000536 1987-09-18 1988-08-29 Plasmides recombinants produisant l'hormone d'adenocorticotropique humaine (acth) WO1989002462A1 (fr)

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DEP3731875.6 1987-09-18
DE19873731875 DE3731875A1 (de) 1987-09-18 1987-09-18 Gentechnische herstellung von neukombinierten plasmiden fuer die produktion von humanem adrenocorticotropen horman (acth) und davon abgeleiteten derivaten durch enzymatische spaltung primaer synthetisierter fusionsproteine

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CA2666887A1 (fr) 2006-10-18 2008-04-24 Research Development Foundation Therapies alpha-msh destinees au traitement de maladies auto-immunes

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Publication number Priority date Publication date Assignee Title
EP0020290A1 (fr) * 1979-05-31 1980-12-10 Schering Aktiengesellschaft Procédé pour scinder spécifiquement des séquences peptidiques des protéines
EP0023882A2 (fr) * 1979-08-03 1981-02-11 Schering Aktiengesellschaft Plasmides vecteurs, plasmides contenant des gènes pour phosphatases alcalines, et bactéries contenant ces derniers, leur préparation et application
US4351901A (en) * 1980-03-24 1982-09-28 Cetus Corporation Method for single nucleotide alteration
EP0131363A1 (fr) * 1983-05-24 1985-01-16 Celltech Limited Produits polypeptidiques et protéiniques et procédés pour leur production et utilisation
EP0213676A2 (fr) * 1985-08-30 1987-03-11 Alfio Bertolini Compositions pharmaceutiques contenant l'ACTH(1-24)pour la thérapie des états de choc et des insuffisances respiratoires et cardiocirculatoires

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0020290A1 (fr) * 1979-05-31 1980-12-10 Schering Aktiengesellschaft Procédé pour scinder spécifiquement des séquences peptidiques des protéines
EP0023882A2 (fr) * 1979-08-03 1981-02-11 Schering Aktiengesellschaft Plasmides vecteurs, plasmides contenant des gènes pour phosphatases alcalines, et bactéries contenant ces derniers, leur préparation et application
US4351901A (en) * 1980-03-24 1982-09-28 Cetus Corporation Method for single nucleotide alteration
EP0131363A1 (fr) * 1983-05-24 1985-01-16 Celltech Limited Produits polypeptidiques et protéiniques et procédés pour leur production et utilisation
EP0213676A2 (fr) * 1985-08-30 1987-03-11 Alfio Bertolini Compositions pharmaceutiques contenant l'ACTH(1-24)pour la thérapie des états de choc et des insuffisances respiratoires et cardiocirculatoires

Non-Patent Citations (1)

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Title
Chemical Abstracts, Band 109, Nr. 13, 26. September 1988, (Columbus, Ohio, US), I.I. Baturina et al.: "Studies on the direct expression of peptide genes in Escherichia coli. I. Chemical-enzymic synthesis and cloning of the adreno-cortocotropic hormone ACTH4-10 genetic equivalent fragment", siehe Seiten 176-177 *

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DE3731875A1 (de) 1989-03-30
JPH03500242A (ja) 1991-01-24

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