EP0861325A1 - Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine - Google Patents

Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine

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Publication number
EP0861325A1
EP0861325A1 EP96938115A EP96938115A EP0861325A1 EP 0861325 A1 EP0861325 A1 EP 0861325A1 EP 96938115 A EP96938115 A EP 96938115A EP 96938115 A EP96938115 A EP 96938115A EP 0861325 A1 EP0861325 A1 EP 0861325A1
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EP
European Patent Office
Prior art keywords
peptide
fusion protein
pth
seq
streptavidin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP96938115A
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German (de)
English (en)
Inventor
Erhard Kopetzki
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Roche Diagnostics GmbH
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Roche Diagnostics GmbH
Boehringer Mannheim GmbH
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Publication of EP0861325A1 publication Critical patent/EP0861325A1/fr
Withdrawn legal-status Critical Current

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    • 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
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • 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
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • 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

  • the invention relates to a process for the recombinant production of peptides by expression of fusion proteins with streptavidin and subsequent enzymatic cleavage of the fusion protein
  • Peptides are usually understood to mean substances which consist of up to about 100 amino acids.
  • the production of such peptides is either chemical (Kent, SBH et al (1988) (I), Hodson, JH, (1993) (2) or recombinant ( Kopetzki, E et al (1994) (3) Winnacker, E -L (1987) (4), Harris, TJR (1983) (5))
  • peptides can be used for the recombinant production of peptides.
  • direct expression in the cytoplasm of microorganisms or cell lines can take place.However, this requires a minimum polypeptide length of approx. 80 to 100 amino acids. Smaller peptides are not stable and are broken down by proteolysis As a rule, an additional N-terminal methionine and the yields are very low
  • the object of the present invention is to provide a method by means of which peptides can be made available via streptavidin fusion proteins in high yield and purity with complete separation from the streptavidin portion
  • the object is achieved according to the invention by a process for the recombinant production of a peptide by expression of a DNA in microorganisms, preferably prokaryotes, which codes for a fusion protein of streptavidin and the peptide mentioned, streptavidin and peptide via a peptide sequence which can be cleaved by an endoproteinase, isolation of the insoluble, inactive fusion protein, solubilization of the inactive fusion protein with a denaturing agent, dilution of the denaturing agent at a pH value between 8.5 and 11 until the fusion protein can be cleaved by an endoproteinase, cleavage of the fusion protein, Lower the pH until the cleaved streptavidin and uncleaved fusion protein precipitate and purify the desired peptide from the supernatant
  • streptavidin fusion proteins can be expressed very well in prokaryotes and can be isolated in the form of insoluble inactive proteins (inclusion bodies). Streptavidin fusion proteins solubilized in denaturing agents can be diluted so far at pH values above 8.5 that they can be digested with an endoproteinase without precessing
  • the fusion protein does not have to be renatured to the active protein.
  • the released streptavidin and possibly not cleaved streptavidin fusion protein can be separated from the desired peptide by precipitation at pH values below 6
  • the method according to the invention is suitable for producing a large number of short-chain peptides.
  • the method is suitable for the production of natriuretic peptides and parathyroid hormone peptides.
  • Natriuretic peptides are peptides with natriuretic activity, which are formed in the cardiac ventricle, the adrenal gland and the brain from a precursor polypeptide (prohormone) and have a ring of 17 amino acids as structural element, which is separated by a disulfide bridge between two Cysteine residues is formed.
  • Precursor polypeptides are e.g. B. the "atriaT natriuretic peptide (ANP 1-126) or cardiodilatin (CCD 1-126) and the" brain "natriuretic peptides of the B and C type.
  • Preferred NP peptides are derived from the" human ⁇ atrial -natriuretic peptide "(h ⁇ ANP).
  • the C-terminal h ⁇ ANP fragments of amino acids 95-126, 99-126 and 102-126 are particularly preferred.
  • Urodilatin (CDD 95-126) is a natriuretic peptide which can be obtained from human urine (Forssmann, K. et al. (1988) (23).
  • the peptide has a length of 32 amino acids and forms a ring from 17 amino acids through the formation of a disulfide bridge between two cysteine residues and belongs to the cardiodilatin / "atriar -natriuretic peptide (CDD / ANP) family.
  • ⁇ -ANP 99-126
  • Urodilatin (CCD 95 - 126) is probably created in vivo by cleavage of this propeptide between amino acids 94 and 95.
  • the approximately 3.5 kDa urodilatin peptide differs from ⁇ -ANP (99 - 126) -Peptide through a 4-amino acid extension at the N-terminus
  • the amino acid sequence and the structure of urodilatin are described, for example, in Drummer, C. et al. (1993) (24)
  • Urodilatin binds to the membrane-bound ANP receptors A and B. and activates an intracellular guanylate cycla coupled to the receptor This causes the formation of the "second messenger" cGMP, which mediates the diuretic and natriuretic effects in the kidney and the relaxing effect on the smooth vascular muscles. (Heim, J.M. (1989) (25)).
  • Urodilatin is thus a preferred therapeutic agent for the prophylaxis and therapy of acute kidney failure, e.g. B. in patients after heart or liver transplants.
  • B. kidney failure
  • Urodilatin is thus a preferred therapeutic agent for the prophylaxis and therapy of acute kidney failure, e.g. B. in patients after heart or liver transplants.
  • parathyroid hormone parathyroid hormone
  • the method according to the invention for producing parathyroid hormone (PTH) and its fragments can also be used advantageously.
  • the DNA and amino acid sequence of PTH is described, for example, in Rokkones, E. et al. (1994) (16).
  • the human parathyroid gene codes for a pre-pro-PTH protein of 115 amino acids. After splitting off the signal sequence and the prosegement, the matured PTH hormone has 84 amino acids (PTH 1 - 84). It has been shown that PTH, which is produced recombinantly in E. coli and S. cerevisiae, is unstable and degrades rapidly.
  • the production of PTH fusion proteins is described by Forsberg, G. et al. (1991) (17).
  • a nucleic acid is produced which codes for a fusion protein from mature PTH (1-84) and a 15 kD IgG binding protein.
  • a cleavage site for thrombin or subtilisin is inserted between the two protein parts.
  • This fusion protein is also unstable and is already degraded to a considerable extent when expressed in E. coli.
  • the degradation of PTH (1-84) could not be prevented even by secretion of the mature PTH (1-84) hormone into the periplasm of E.coli using the protein A signal sequence.
  • the half-life of PTH (1 - 84) in E. coli is only a few minutes.
  • the fusion proteins can be cleaved enzymatically with a specifically cleaving proteinase (restriction proteinase).
  • the proteinase is selected taking into account the amino acid sequence of the peptide to be produced. Care should be taken to ensure that the recognition / cleavage sequence of the restriction proteinase does not occur in the desired peptide and preferably not in the carrier portion (streptavidin portion) of the fusion protein, ie it should only occur once in the cleavage region (linker region).
  • cleaving endoproteinases z. B.
  • enterokinase factor Xa, thrombin, subtilisin BPN variants / ubiquitin protein peptidase, renin, collagenase, trypsin, chymotrypsin, endoproteinase Lys-C, Kallekrein (Carter, P., (12)), TEV proteinase (Parks, TD et al ., Anal. Biochem. 216 (1994) 413-417) (36), IgA proteinase (Pohlner, J. et al., Nature 325 (1987) 458-462) (37), Kex2p proteinase (EP-A 0467 839) (38) or S. aureus V8 proteinase.
  • Endoproteinase LysC which specifically cleaves proteins and peptides at the C-terminal end of lysine, is preferably used.
  • Such an enzyme is known, for example, from fungi or bacteria (DE 30 34 045 C2).
  • Endoproteinase LysC is particularly well suited for the production of peptides that do not contain a lysine residue, such as. B. Urodilatin.
  • a peptide sequence which can be cleaved by an endoproteinase is a short-chain peptide sequence which preferably consists of 5-15 amino acids and which contains a cleavage site for the desired endoproteinase at the C-terminal.
  • This N-terminal linker preferably contains, in addition to the desired endoproteinase recognition sequence, a combination of several amino acids, selected from the amino acids Gly, Thr, Ser, Ala, Pro, Asp, Glu, Arg and Lys.
  • a linker is particularly preferably used in which 2-8 of these additional amino acids are the negatively charged amino acids Asp and / or Glu.
  • Streptavidin for example, as described in EP-B 0 198 015 (7) and EP-A 0 612 325 (8), can be used as streptavidin.
  • Other streptavidin derivatives or fragments as described for example by Sano, T. et al., (9), are also suitable.
  • a streptavidin which is truncated (shortened) at the N-terminus and / or C-terminus is preferably used. This prevents aggregation and proteolysis (Sano, T. et al., (9)).
  • a streptavidin is preferably used which begins with amino acids 10-20 and ends with amino acids 130-140 (numbering analogously: Argarana, C.E. et al. (1986) (33)).
  • a streptavidin of amino acids 16-133 or 13-139 is particularly preferably used.
  • the fusion proteins are produced by expressing a DNA (nucleic acid sequence) which codes for the fusion protein in microorganisms, preferably in prokaryotes.
  • a DNA nucleic acid sequence
  • the expression vector used should not contain any elements which mediate secretion of the protein into the medium.
  • a DNA suitable for expression can preferably be produced synthetically. Such methods are known to the person skilled in the art common and described, for example, in Beattie, KL and Fowler, RF (1991) (34), EP-B 0 424 990 (35), Itakura, K et al (1977) (20).
  • the nucleic acid sequence of the proteins according to the invention can be appropriately modified Such modifications are, for example
  • Fusion proteins can be found both in prokaryotes and in other cells, for example in eukaryotic host cells such as yeasts (for example Saccharomyces, Pichia, Hansenula and Kluyveromyces) and fungi such as Aspergillis and Trichoderma in the form of insoluble protein aggregates, so-called "inclusion bodies" (IBs ), Inclusion bodies occur when the rate of synthesis of the protein in the cell is greater than the rate of folding to the active native protein. In this case, the protein aggregates in the cell, preferably in the cytoplasm. There the protein becomes denatured, compressed and insoluble Form deposited in the cell. As a result, the cell experiences as little interference as possible with its other cell functions
  • Suitable prokaryotic host organisms are, for example, Escherichia, Streptomyces or Bacillus.
  • the microorganisms preferably prokaryotes, are transformed in the usual way with the vector which contains the DNA coding for the fusion protein, and then fermented in the usual manner.
  • the insoluble, inactive protein (IBs) is isolated from the cells in a conventional manner, for example by centrifugation (pellet fraction). Protein aggregates can be obtained by washing the pellet with z.
  • B. detergent-containing buffers are further enriched.
  • the IBs are treated with a denaturing agent, e.g. Guanidine hydrochloride, urea or a urea derivative (see, for example, US Pat. No. 5,453,363) is solubilized and transferred to a suitable non-denaturing buffer (pH> 8.5) by dilution or dialysis.
  • a denaturing agent e.g. Guanidine hydrochloride, urea or a urea derivative (see, for example, US Pat. No. 5,453,363) is solubilized and transferred to a suitable non-denaturing buffer (pH> 8.5) by dilution or dialysis.
  • the dilution is carried out in such a way that the remaining denaturing agent does not significantly affect the enzymatic hydrolysis of the fusion protein.
  • the dilution is preferably carried out in a pulsed manner, for example by dropping the IB solubilizate in buffer (pH> 8.5) which contains no denaturing agent.
  • Such a pulse-like dilution enables a practically simultaneous removal of the effect of the denaturing agent and separation of the molecules to be solubilized. This largely avoids an undesired intermolecular interaction (aggregation) of the molecules to be solubilized.
  • the fusion proteins produced by the process according to the invention are not broken down in the host cells and can be completely cleaved enzymatically without any significant cleavage taking place in the peptide portion itself (for example PTH or urodilatin).
  • the method according to the invention is particularly suitable for the production of urodilatin, parathyroid hormone and their fragments.
  • the urodilatin fragments of amino acids 95-126, 99-126 or 102-126 and the parathyroid fragment of amino acids 1-37 are particularly preferably produced.
  • Example 1 shows the DNA segments A and B obtained according to Example 1.
  • the expression vector for the core-SA-URO (95-126) fusion gene with endoproteinase LysC cleavage site is based on the expression vector pSAM-CORE for core streptavidin.
  • the preparation and description of the plasmid pSAM-CORE is described in WO 93/09144 (11).
  • the unique Nhel restriction site located at the 3 'end in front of the stop codon of the core-SA gene was used to construct core-SA fusion proteins.
  • GGCCGCATGGACCGTATCGGTGCTCAGTCCGGACTGGGTTGCAACTCCTTCCGTT ACTAATGA SEQ ID NO: 5
  • DNA segment B (FIG. 1) "aged” (reaction buffer: 12.5 mmol / l Tris-HCl, pH 7.0 and 12.5 mmol / l MgCl2; oligonucleotide concentration: 1 pmol / 60 ⁇ l in each case ) and the hybridization products A and B each subcloned into the polylinker region of the E. coli pUCBM21 vector (Boehringer Mannheim GmbH, Mannheim, Germany) (DNA segment A, interfaces: EcoRI and Notl; DNA segment B, interfaces: Notl and Hindill). The DNA sequence of the two subcloned DNA segments was confirmed by means of DNA sequencing.
  • the expression plasmid pSA-EK-URO for the core-SA-URO (95-126) fusion gene was then used in a three-fragment ligation from the Nhe / Notl-DNA segment A, the Notl / Hindlll-DNA segment B and the approx. 2.9 kBp long Nhel / Hindlll-pSAM-CORE vector fragment composed. After double digestion, the DNA segments A and B were isolated with the corresponding endonucleases from the corresponding pUCBM21 plasmid derivatives. The desired plasmid pSA-EK-URO was identified by restriction mapping and the DNA sequence of the linker-urodilatin region was checked again by DNA sequencing.
  • PTH Parathormone fragment of amino acids 1-37, amino acid sequence described in Handy, G.N. et al., Proc. Natl. Acad. Be. USA 78 (1981) 7365-7369 (39).
  • the vector pSA-EK-PTH for expressing the core-SA-PT ⁇ (1-37) fusion gene with enterokinase cleavage site was produced according to the strategy described in Example 1 for the core-SA-URO (95-126) fusion gene with enterokinase cleavage site.
  • the DNA sequence of the two subcloned DNA segments was confirmed by means of DNA sequencing.
  • the expression plasmid pSA was then confirmed - EK-PT ⁇ for the core-SA-EK-PTH (l-37) fusion gene in a three-fragment ligation from the Nhel / NcoI-DNA segment C, the NcoI / HindüI-DNA segment D and the approx. 2.9 kBp long Nhel / Hindlll-pSAM-CORE vector fragment composed.
  • the DNA segments C and D were isolated after double digestion with the corresponding endonucleases from the corresponding pUCBM21 plasmid derivatives.
  • the desired plasmid pSA-EK-PTH was identified by restriction mapping and the DNA sequence of the enterokina elinker PTH region checked again by DNA sequencing
  • the plasmid pSA-THRO-PTH is derived from the core-SA-EK-PTH expression plasmid pSA-EK-PTH (see Example 2) by replacing the coding region for the enterokinase linker with a thrombin linker
  • the amino acid sequence of the thrombin linker [GDFLAEGLVPR] used (SEQ ID NO 15) is based on the natural thrombin cleavage site in fibrinogen (amino acid position 6-16) and the minimal recognition sequence for thrombin (Carter, P In Ladisch, MR, Willson, RC, Painton, CC, Builder, SE eds (1990) (12))
  • the plasmid pSA-EK-PTH was digested with Nhel and PvuII, the approximately 2.9 kbp long Nhel / PvuII-pSA-EK-PTH vector fragment was isolated and with that from the 2 complementary oligonucleotides 9 (SEQ ID NO 11) and 10 (SEQ ID NO 12) DNA segment E (FIG. 3) produced by hybridization
  • the desired plasmid construction pSA-THRO-PTH was identified by restriction mapping and the exchanged left region was checked by DNA sequencing.
  • the plasmid pSA-TEV-PTH is derived from the core-SA-EK-PTH expression plasmid pSA-EK-PTH (see Example 2) by replacing the coding region for the enterokinase linker with a TEV linker.
  • the plant virus TEV NIa proteinase ("tobacco etch virus”) recognizes the amino acid sequence ENLYFQiG / S and cleaves between gin and gly or ser (Dougherty, W.G. et al., (1988)) (13).
  • tobacco etch virus recognizes the amino acid sequence ENLYFQiG / S and cleaves between gin and gly or ser (Dougherty, W.G. et al., (1988)) (13).
  • the recombinantly produced enzyme was obtained from GIBCO BRL (Life Technologies, Ine Gaithersburg, MD, USA).
  • the plasmid pSA-EK-PTH was digested with Nhel and PvuII, the approx. 2.9 kbp long Nhel / PvuII-pSA-EK-PTH vector fragment was isolated and with that from the 2 complementary oligonucleotides JI (SEQ ID NO: 13) and ⁇ 2 (SEQ ID NO: 14) DNA segment F (FIG. 3) produced by hybridization.
  • the E. coli Kl 2 strain RM82 (a methionine revertant from ED 8654, Murray, NE et al. (1977)) (14) was in each case with one of the expression plasmids pSA described in Examples 1-4 -EK-URO, pSA-EK-PTH, pSA-THRO-PTH and pSA-TEV-PTH (ampicillin resistance) and the lacW repressor plasmid pUBS500 (kanamycin resistance, preparation and description see: EP-A 0368342) transformed.
  • the RM82 / pUBS500 / pSA-EK-URO, RM82 / pUBS500 / pSA-EK-PTH, RM82 / pUBS500 / pSA-THRO-PTH and RM82 / pUBS500 / pSA-TEV-PTH cells were in DYT medium (1% ( w / v) yeast extract, 1% (w / v) Bacto Tryptone (Difco, Detroit, USA) and 0.5% NaCl), with 50 mg / l ampicillin and 50 mg / l kanamycin up to an optical density at 550 nm attracted from 0.6-0.9 and then induced with IPTG (isopropyl- ⁇ -D-thiogalactoside) (1-5 mmol / l final concentration). After an induction phase of 4-8 hours, the cells were harvested by centrifugation and the cell pellets were washed with 25 mmol / l potassium phosphate buffer
  • the cell pellets each from 1 ml of centrifuged growth medium (RM82 / pUBS500 / pSA-EK-URO, RM82 / pUBS500 / pSA-EK-PTH, RM82 / pUBS500 / pSA-THRO-PTH and
  • RM82 / ⁇ UBS500 / pSA-TEV-PTH cells were resuspended in 0.25 ml 10 mmol / l phosphate buffer, pH 6.8 and 1 mmol / l EDTA and the cells were disrupted by ultrasound treatment. After centrifugation, 1/5 volume of 5xSDS sample buffer (1xSDS sample buffer: 50 mmol / l Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) was added to the supernatant.
  • 5xSDS sample buffer 50 mmol / l Tris-HCl, pH 6.8, 1% SDS, 1% mercaptoethanol, 10% glycerol, 0.001% bromophenol blue
  • the insoluble cell debris fraction was resuspended in 0.3 ml of lxSDS sample buffer with 6-8 M urea, the samples were incubated for 5 minutes at 95 ° C. and centrifuged. The proteins were then separated by SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli, UK (1970)) (15) and stained with Coomassie Brilliant Blue R dye.
  • the core-SA fusion proteins synthesized in E. coli were homogeneous and were found exclusively in the insoluble cell debris fraction (IBs). The expression level for the core-SA fusion proteins was 30-50% based on the total E. coli protein
  • E coli RM82 / pUBS500 / pSA-EK-URO, RM82 / pUBS500 / pSA-EK-PTH, RM82 / pUBS500 / pSA-THRO-PTH and RM82 / pUBS500 / pSA-TEV-PTH cells were in 1 1 0, 1 mol / 1 Tris-HCl, pH 7.0 suspended at 0 ° C, 300 mg of lysozyme added and incubated for 20 minutes at 0 ° C.
  • the cells were then mechanically disrupted by means of high pressure dispersion and the DNA by addition of 2 ml 1 mol / 1 MgCl2 and 10 mg DNAse (Boehringer Mannheim # 154709) digested in 30 minutes at 25 ° C. Then 500 ml 60 mmol / l EDTA, 6% Triton® XI 00 and 1.5 mol / 1 NaCl, pH 7.0 was added and incubated for a further 30 minutes at 0 ° C. The insoluble constituents (cell dummies and IBs) were then sedimented by centrifugation
  • the pellet was suspended in 1 1 0.1 mol / 1 Tris-HCl, 20 mmol / l EDTA, pH 6.5, incubated for 30 minutes at 25 ° C. and the IB preparation was isolated by centrifugation
  • IB pellet 25 g were suspended in 200 ml of 10 mmol / l Tris-HCl buffer, 8 mol / 1 urea, 10 mmol / l EDTA, pH 7.0 by stirring for 2 hours at 25 ° C. The insoluble constituents were separated by centrifugation and the clear supernatant processed further
  • the core-SA fusion proteins with enterokinase interface were in a concentration of 0.3 to 3 mg / ml and a substrate / proteinase ratio of 1:20 to 1: 250 (enterokinase, restriction proteinase from calf intestine, Boehringer Mannheim, Mannheim, Germany) in 50 mmol / l Tris-HCl, pH 8.0 digested at 30 ° C. and the time course of the enzymatic cleavage (kinetics) was analyzed by analytical reversed phase HPLC (see Example 9.1). For this purpose, samples (10 to 100 ⁇ l) were taken at intervals of 1 to 3 hours from the reaction mixture over a period of 6 to 24 hours.
  • LysC endoproteinase cleavage (Lys cleavage site)
  • the core SA-EK-URO fusion protein was obtained in a concentration of 0.3 to 3 mg / ml and a substrate / proteinase ratio of 1: 1000 to 1: 25000 (LysC endoproteinase from Lysobacter enzymogenes, sequencing grade; Boehringer Mannheim, Mannheim , Germany) in 50 mmol / l Tris-HCl, pH 8.0 at 30 to 35 ° C and the time course of the enzymatic cleavage was analyzed by analytical reversed phase HPLC (see Example 9.1). For this purpose, samples (10 to 100 ⁇ l) were taken at intervals of 1 to 3 hours from the reaction mixture over a period of 6 to 24 hours.
  • the core-SA-THRO-PTH fusion protein was in a concentration of 0.3 to 3 mg / ml and a substrate / proteinase ratio of 1:50 to 1: 500 (thrombin from human plasma, Boehringer Mannheim, Mannheim, Germany) in 50 mmol / l Tris-HCl, pH 8.8 digested at 25 to 30 ° C and the time course of the enzymatic cleavage was analyzed by analytical reversed phase HPLC (see Example 9.1). For this purpose, samples (10 to 100 ⁇ l) were taken at intervals of 1 to 3 hours from the reaction mixture over a period of 6 to 24 hours. TEV NIa proteinase cleavage (GluAsnLeuTyrPheGln-i-Gly / Ser cleavage sequence)
  • the core-SA-TEV-PTH fusion protein was obtained in a concentration of 0.3 to 3 mg / ml and a substrate / proteinase ratio of 1:50 to 1: 500 (recombinant TEV NIa restriction proteinase, GIBCO BRL Life Technologies, Inc. Gaithersburg , MD, USA) in 50 mmol / l Tris-HCl, pH 8.0, 0.5 mmol / l EDTA and 1 mmol / l DTT at 30 ° C and the time course of the enzymatic cleavage by analytical reversed phase HPLC ( see example 9.1).
  • the reaction mixture was used over a period of 6 to
  • the released core-SA carrier protein and uncleaved core-SA fusion protein were precipitated from the cleavage mixture by lowering the pH (pH ⁇ 6).
  • the cleavage mixture was treated with 1 mol / 1 citric acid to a final concentration of
  • the enzymatically released peptides can be further purified using chromatographic methods known to the person skilled in the art.
  • the bound material was eluted by a gradient of 0 to 1 mmol / l NaCl in equilibration buffer (10 up to 20 SV, 1 SV / hour). 10.2 Purification of the peptides by reversed phase HPLC
  • the sample volume was 10 - 100 ⁇ l, corresponding to 1 100 ⁇ g protein
  • the detection was carried out with a UV detector at 220 nm. Chromatography was carried out at a flow rate of 0.5 ml / min.
  • the identity and purity of the purified peptides was determined by mass spectroscopy (PD-MS and laser desorption spectroscopy), analytical reversed phase HPLC, isoelectric focusing (Bark, JE et al., J. Forensic Sei. Soc. 16 (1976) 115-120 ( 42), SDS PAGE (Laemmli, UK, Nature 227 (1970) 680-685 (43)) and capillary electrophoresis, compared with a chemically produced standard.

Abstract

L'invention concerne un procédé de préparation par recombinaison de peptides par expression d'un ADN dans des micro-organismes, qui code une protéine de fusion issue de la streptavidine et d'un desdits peptides. La streptavidine et le peptide sont liés par une séquence peptidique pouvant être clivée par une endoprotéase. Ce procédé comprend également l'isolation de la protéine inactive non soluble, la solubilisation de la protéine inactive avec un dénaturant, la dilution du dénaturant à un pH compris entre 8,5 et 11, jusqu'à ce que le clivage de la protéine de fusion par une endoprotéinase puisse s'effectuer, le clivage de la protéine de fusion, abaissement du pH, jusqu'à ce que la strepavidine et la protéine de fusion clivée forment un précipité, et la purification du peptide voulu à partir du surnageant. Ce procédé s'utilise notamment pour préparer de la parathormone et de l'urodilatine, ainsi que des fragments de celles-ci.
EP96938115A 1995-11-16 1996-11-06 Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine Withdrawn EP0861325A1 (fr)

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DE19542702 1995-11-16
DE19542702 1995-11-16
PCT/EP1996/004850 WO1997018314A1 (fr) 1995-11-16 1996-11-06 Procede de preparation de peptides par l'intermediaire de proteines de fusion issues de la streptavidine

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EP0861325A1 true EP0861325A1 (fr) 1998-09-02

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EP (1) EP0861325A1 (fr)
JP (1) JP2000500019A (fr)
AU (1) AU7566396A (fr)
CA (1) CA2237296C (fr)
WO (1) WO1997018314A1 (fr)

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WO1997018314A1 (fr) 1997-05-22
US6136564A (en) 2000-10-24
CA2237296C (fr) 2008-10-07
CA2237296A1 (fr) 1997-05-22
JP2000500019A (ja) 2000-01-11
AU7566396A (en) 1997-06-05

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