EP0861325A1 - Process for the preparation of peptides by way of streptavidin fusion proteins - Google Patents
Process for the preparation of peptides by way of streptavidin fusion proteinsInfo
- 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
- Authority
- 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.)
- Withdrawn
Links
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Classifications
-
- 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, i.e. 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.
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Abstract
The invention relates to a process for recombinant preparation of peptides by expression of a DNA in micro-organisms, which DNA codes for a fusion protein made of streptavidin and one of the said peptides. Streptavidin and the peptide are bound by a peptide sequence which can be cleaved by an endoproteinase. The process also includes isolation of the insoluble, inactive protein, solublisation of the inactive protein using a denaturant, dilution of the denaturant at a pH value of between 8.5 and 11 until cleaving of the fusion protein by an endoproteinase can take place, cleaving of the fusion protein, lowering of the pH value until streptavin and non-cleaved fusion protein precipitate, and cleaning of the desired peptide from the supernatant. Said process is particularly suitably for producing parathromone and urodilatin and fragments thereof.
Description
Verfahren zur Herstellung von Peptiden über Streptavidin-Fusionsproteine Process for the preparation of peptides via streptavidin fusion proteins
Gegenstand der Erfindung ist ein Verfahren zur rekombinanten Herstellung von Peptiden durch Expression von Fusionsproteinen mit Streptavidin und anschließender enzymatischer Spaltung des FusionsproteinsThe 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
Unter Peptiden werden üblicherweise Substanzen verstanden, die aus bis zu ca 100 Ami¬ nosäuren bestehen Die Herstellung solcher Peptide ist entweder chemisch (Kent, S B H et al (1988) (I), Hodson, J H , (1993) (2) oder rekombinant (Kopetzki, E et al (1994) (3) Winnacker, E -L (1987) (4), Harris, T J R (1983) (5)) möglichPeptides 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))
Die Nachteile der chemischen Peptidsynthese liegen insbesondere darin, daß eine ökonomische Synthese lediglich bis ca 30 bis 40 Aminosauren möglich ist und sich häufig bei der Synthese unerwünschte Modifizierungen (Fehlsequenzen, nicht abgespaltene Schutzgruppen) bilden Weitere Probleme sind die Racemisierung bei Fragmentkopplung, Schwierigkeiten bei der Abspaltung von Schutzgruppen und schließlich die aufwendige ReinigungThe disadvantages of chemical peptide synthesis are, in particular, that economical synthesis is only possible up to approximately 30 to 40 amino acids and that undesirable modifications (incorrect sequences, protective groups which have not been split off) frequently form during synthesis. Further problems are racemization in the case of fragment coupling, difficulties in splitting off of protecting groups and finally the elaborate cleaning
Zur rekombinanten Herstellung von Peptiden können verschiedene Verfahren angewendet werden Beispielsweise kann eine direkte Expression im Cytoplasma von Mikroorganismen oder Zellinien erfolgen Hierfür ist jedoch eine Mindestpolypeptidlange von ca 80 bis 100 Aminosauren erforderlich Kleinere Peptide sind nicht stabil und werden durch Proteolyse abgebaut Zudem enthalten diese Proteine in der Regel ein zusatzliches N-terminales Methio¬ nin, und die Ausbeuten sind sehr geringVarious methods can be used for the recombinant production of peptides.For example, 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
Durch Expression loslicher Fusionsproteine mit selektiver Spaltsequenz und anschließender Freisetzung des gewünschten Peptids durch chemische oder enzymatische Spaltung, kann die Herstellung solcher Peptide verbessert werden (Itakura, K et al (1977) (20), EP-B 0 001 930 (21), Gram, H et al (1994) (19), Sharma, A. et al (1992) (22)) Der Nachteil von loslichen Fusionsproteinen ist aber insbesondere, daß sie vorwiegend im nicht strukturierten Peptidbe- reich durch Proteolyse in der Zelle bzw wahrend der Sekretion und Aufarbeitung degradiert werden können
Die Herstellung von Streptavidin-Fusionsproteinen ist in der EP-B 0 198 015, bei Sano, T (1991) (9) und Sano, T (1992) (10) beschrieben Solche chimare Proteine umfassen bei Sano als Streptavidinanteil die Aminosäuren 16 - 133 von Streptavidin, einen Polylinker und die Sequenz des "target-Proteins". Als target-Proteine sind von Sano das Maus Metallothionein I Protein und das T7-Gen-10-Protein beschrieben Diese chimaren Proteine enthielten jedoch keine Spaltstelle, über die das "target-Protein" vom Streptavidin-Anteil wieder abgespalten werden kann In der EP-B 0 198 015 wird die Herstellung von Streptavidiniusionsproteinen in Streptomyces beschrieben. Die Isolierung des Peptidanteils aus dem Fusionsprotein ist jedoch sehr aufwendig So muß beispielsweise vor und nach der Spaltung jeweils eine Affinitätschro¬ matographie mit Iminobiotin als Ligand durchgeführt werdenThe production of such peptides can be improved by expressing soluble fusion proteins with a selective cleavage sequence and subsequent release of the desired peptide by chemical or enzymatic cleavage (Itakura, K et al (1977) (20), EP-B 0 001 930 (21), Gram , H et al (1994) (19), Sharma, A. et al (1992) (22)) However, the disadvantage of soluble fusion proteins is in particular that they are predominantly in the unstructured peptide range by proteolysis in the cell or during the Secretion and workup can be degraded The production of streptavidin fusion proteins is described in EP-B 0 198 015, in Sano, T (1991) (9) and Sano, T (1992) (10). In Sano, such chimeric proteins comprise amino acids 16-133 as streptavidin portion of streptavidin, a polylinker and the sequence of the "target protein". Sano describes the mouse metallothionein I protein and the T7 gene 10 protein as target proteins. However, these chimeric proteins did not contain any cleavage point via which the "target protein" can be cleaved off from the streptavidin portion. B 0 198 015 describes the production of streptavidinius ion proteins in Streptomyces. However, the isolation of the peptide portion from the fusion protein is very complex. For example, affinity chromatography with iminobiotin as the ligand must be carried out before and after the cleavage
Die Aufgabe der vorliegenden Erfindung besteht darin, ein Verfahren bereitzustellen, mit dem Peptide über Streptavidin-Fusionsproteine in hoher Ausbeute und Reinheit bei möglichst voll- standiger Abtrennung vom Streptavidinanteil zur Verfugung gestellt werden könnenThe 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
Die Aufgabe wird erfindungsgemäß gelost durch ein Verfahren zur rekombinanten Herstellung eines Peptids durch Expression einer DNA in Mikroorganismen, vorzugsweise Prokaryonten, welche für ein Fusionsprotein aus Streptavidin und dem genannten Peptid codiert, wobei Streptavidin und Peptid über eine Peptidsequenz, welche durch eine Endoproteinase spaltbar ist, verbunden sind, Isolierung des unlöslichen, inaktiven Fusionsproteins, Solubilisierung des inaktiven Fusionsproteins mit einem Denaturierungsmittel, Verdünnung des Denaturierungs- mittels bei einem pH-Wert zwischen 8,5 und 11, bis die Spaltung des Fusionsproteins durch eine Endoproteinase durchführbar ist, Spaltung des Fusionsproteins, Erniedrigung des pH-Werts, bis das abgespaltene Streptavidin und nicht gespaltenes Fusionsprotein prazipitie- ren, und Reinigung des gewünschten Peptids aus dem ÜberstandThe 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
Das erfindungsgemaße Verfahren nutzt den Vorteil, daß Streptavidin-Fusionsproteine in Prokaryonten sehr gut exprimierbar sind und sich in Form von unlöslichen inaktiven Proteinen (inclusion bodies) isolieren lassen In Denaturierungsmitteln solubilisierte Streptavidin- Fusionsproteine lassen sich bei pH-Werten über 8,5 so weit verdünnen, daß sie, ohne zu prazipitieren, mit einer Endoproteinase verdaut werden könnenThe method according to the invention takes advantage of the fact that 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
Ein weiterer wesentlicher Vorteil des erfindungsgemäßen Verfahrens liegt darin, daß eine Naturierung des Fusionsproteins zum aktiven Protein nicht erforderlich ist Schließlich können das freigesetzte Streptavidin und gegebenenfalls nicht gespaltenes Streptavidin-Fusionsprotein durch Prazipitation bei pH- Werten unter 6 vom gewünschten Peptid abgetrennt werden Das
erfindungsgemäße Verfahren ist zur Herstellung einer Vielzahl von kurzkettigen Peptiden geeignet. Insbesondere ist das Verfahren zur Herstellung von natriuretischen Peptiden und Parathormonpeptiden geeignet.Another important advantage of the method according to the invention is that the fusion protein does not have to be renatured to the active protein. Finally, 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. In particular, the method is suitable for the production of natriuretic peptides and parathyroid hormone peptides.
Natriuretische Peptide (NP-Peptide) sind Peptide mit natriuretischer Aktivität, die im Herz- Ventrikel, der Nebenniere und dem Gehirn aus einem Precursorpolypeptid (Prohormon) gebil¬ det werden und als Strukturelement einen Ring aus 17 Aminosäuren aufweisen, der durch eine Disulfidbrϋcke zwischen zwei Cysteinresten ausgebildet wird. Precursorpolypeptide sind z. B. das "atriaT-natriuretische Peptid (ANP 1 - 126) oder Cardiodilatin (CCD 1 - 126) und die "brain"-natriuretischen Peptide vom B- und C-Typ. Bevorzugte NP-Peptide leiten sich von dem "human α atrial-natriuretic peptide" (hαANP) ab. Besonders bevorzugt sind dabei die C-terminalen hαANP-Fragmente der Aminosäuren 95 - 126, 99 - 126 und 102 - 126.Natriuretic peptides (NP 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 α atrial -natriuretic peptide "(hαANP). The C-terminal hαANP fragments of amino acids 95-126, 99-126 and 102-126 are particularly preferred.
Urodilatin (CDD 95 - 126) ist ein natriuretisches Peptid, welches aus humanem Urin gewon¬ nen werden kann (Forssmann, K. et al. (1988) (23). Das Peptid hat eine Länge von 32 Ami¬ nosäuren, bildet einen Ring aus 17 Aminosäuren durch die Ausbildung einer Disulfidbrücke zwischen zwei Cysteinresten und gehört zu der Cardiodilatin/"atriar -natriuretischen Peptid (CDD/ANP)-Familie. Es entsteht, ebenso wie α-ANP (99 - 126), aus dem ANP-Propeptid (ANP 1 - 126). Urodilatin (CCD 95 - 126) entsteht in vivo vermutlich durch Spaltung dieses Propeptids zwischen den Aminosäuren 94 und 95. Das ca. 3,5 kDa schwere Urodilatin-Peptid unterscheidet sich vom α-ANP (99 - 126)-Peptid durch eine 4- Aminosäuren- Verlängerung am N-Terminus. Die Aminosäuresequenz und die Struktur von Urodilatin sind beispielsweise in Drummer, C. et al. (1993) (24) beschrieben. Urodilatin bindet an die membranständigen ANP-Rezeptoren A und B und aktiviert eine an den Rezeptor gekoppelte intrazelluläre Guanylatcyklase. Dies bewirkt die Bildung des "second messengers" cGMP, der die diureti- schen und natriuretischen Wirkungen in der Niere und die relaxierende Wirkung auf die glatte Gefäßmuskulatur vermittelt. (Heim, J.M. (1989) (25)). Damit ist Urodilatin ein bevorzugtes Therapeutikum zur Prophylaxe und Therapie des akuten Nierenversagens, z. B. bei Patienten nach Herz- oder Lebertransplantationen. (Bub, A. et al. (1992) (26), Drummer, C. et al. (1991) (27) und (1992) (28); Emmeluth, C. et al. (1992) (29); Goetz, K.L. et al. (1990) (30)).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. Like α-ANP (99-126), it arises from the ANP propeptide ( ANP 1 - 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 α-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. (Bub, A. et al. (1992) (26), Drummer, C. et al. (1991) (27) and (1992) (28); Emmeluth, C. et al. (1992) (29); Goetz, KL et al. (1990) (30)).
Ebenfalls vorteilhaft kann das erfindungsgemäße Verfahren zur Herstellung von Parathormon (PTH) sowie dessen Fragmente verwendet werden. Die DNA- und Aminosäuresequenz von PTH ist beispielsweise in Rokkones, E. et al. (1994) (16) beschrieben. Das humane Parathor¬ mongen codiert für ein Pre-Pro-PTH Protein von 115 Aminosäuren. Nach Abspaltung der Signalsequenz und des Prosegments besitzt das gereifte PTH Hormon 84 Aminosäuren (PTH
1 - 84). Es hat sich gezeigt, daß PTH, welches rekombinant in E.coli und S. cerevisiae hergestellt wird, instabil ist und rasch abgebaut wird. Die Herstellung von PTH-Fusionspro- teinen ist von Forsberg, G. et al. (1991) (17) beschrieben. Dazu wird eine Nukleinsäure hergestellt, welche für ein Fusionsprotein aus reifem PTH (1 - 84) und ein 15 kD schweres IgG-Bindeprotein codiert. Zwischen beide Proteinanteile ist eine Spaltstelle für Thrombin oder Subtilisin eingefügt. Auch dieses Fusionsprotein ist instabil und wird bei der Expression in E.coli bereits zu einem beträchtlichen Teil abgebaut. Auch durch Sekretion des reifen PTH (1 - 84) Hormones ins Periplasma von E.coli unter Verwendung der Protein A-Signalsequenz konnte die Degradation von PTH (1 - 84) nicht verhindert werden. Die Halbwertszeit von PTH (1 - 84) in E.coli beträgt nur wenige Minuten.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). For this purpose, 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.
Von Gardella, T.J. et al (1990) (18) wird die Herstellung von PTH (1 - 84) in E.coli über ein mit Faktor Xa spaltbares Fusionsprotein beschrieben. Die Spaltung mit Faktor Xa ist jedoch sehr unvollständig (nach zwei Stunden ca. 50 % Spaltung) oder führt bei längerer Inkubation mit Faktor Xa ebenfalls zum Abbau von PTH (1 - 84).By Gardella, T.J. et al (1990) (18) describes the production of PTH (1-84) in E. coli using a fusion protein which can be cleaved with factor Xa. However, the factor Xa cleavage is very incomplete (approx. 50% cleavage after two hours) or, when incubated with factor Xa for a long time, also leads to the breakdown of PTH (1-84).
Von Gram, H. et al. (1994) (19) wird ebenfalls die Herstellung eines PTH-Fragments (PTH 1 - 38) mittels eines Fusionsproteins in E.coli beschrieben. Zur Abspaltung des C-terminal fusionierten PTH (1 - 38) Peptids wurde ein Asp-Pro-Pro-Linker benutzt. Dieser läßt sich mittels eines 2- Stufenprozesses spalten/entfernen. In einer ersten Reaktion wird die säurelabile Asp-Pro-Peptidbindung chemisch hydrolysiert (Inkubation des Fusionsproteins in 60 mM HCl für 24 Std. bei 50°C). Danach wird in einer zweiten Reaktion das N-terminal verbleibende Pro-Pro-Dipeptid enzymatisch mit Dipeptidylpeptidase IV aus L. lactis entfernt. Dieses Verfahren ist jedoch zeitaufwendig und bedingt durch die saure Hydrolyse werden in beträcht¬ lichem Umfang Nebenprodukte gebildet.By Gram, H. et al. (1994) (19) also describes the production of a PTH fragment (PTH 1-38) by means of a fusion protein in E. coli. An Asp-Pro-Pro linker was used to cleave the C-terminally fused PTH (1-38) peptide. This can be split / removed using a 2-step process. In a first reaction, the acid-labile Asp-Pro peptide bond is hydrolyzed chemically (incubation of the fusion protein in 60 mM HCl for 24 hours at 50 ° C). The N-terminally remaining pro-pro-dipeptide is then enzymatically removed from L. lactis with Dipeptidylpeptidase IV in a second reaction. However, this process is time-consuming and, as a result of the acid hydrolysis, by-products are formed to a considerable extent.
Die Spaltung der Fusionsproteine kann enzymatisch mit einer spezifisch spaltenden Proteinase (Restriktionsproteinase) erfolgen. Die Auswahl der Proteinase erfolgt unter Berücksichtigung der Aminosäuresequenz des herzustellenden Peptids. Es ist darauf zu achten, daß die Erken- nungs-/Spaltsequenz der Restriktionsproteinase möglichst nicht in dem gewünschten Peptid und vorzugsweise auch nicht im Carrieranteil (Streptavidinanteil) des Fusionsproteins vorkommt, d.h. sie sollte nur einmal in der Spaltungsregion (Linkerregion) vorkommen. Als spezifisch spaltende Endoproteinasen sind z. B. Enterokinase, Faktor Xa, Thrombin, Subtilisin BPN Varianten / Ubiquitin Protein Peptidase, Renin, Collagenase, Trypsin, Chymotrypsin, Endoproteinase Lys-C, Kallekrein (Carter, P., (12)), TEV Proteinase (Parks, T.D. 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) oder S. aureus V8 Proteinase geeignet.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). As specifically 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.
Vorzugsweise wird Endoproteinase LysC, welche spezifisch am C-terminalen Ende von Lysin Proteine und Peptide spaltet, verwendet. Ein solches Enzym ist beispielsweise aus Pilzen oder Bakterien (DE 30 34 045 C2) bekannt. Endoproteinase LysC eignet sich besonders gut zur Herstellung von Peptiden, die keinen Lysinrest enthalten, wie z. B. Urodilatin.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.
Unter einer Peptidsequenz, welche durch eine Endoproteinase spaltbar ist, ist im Sinne der vorliegenden Erfindung eine kurzkettige Peptidsequenz zu verstehen, welche vorzugsweise aus 5 - 15 Aminosäuren besteht und die eine Spaltstelle für die gewünschte Endoproteinase C- terminal enthält. Vorzugsweise enthält dieser Linker N-terminal von der gewünschten Endo- proteinaseerkennungssequenz zusätzlich eine Kombination aus mehreren Aminosäuren, aus¬ gewählt aus den Aminosäuren Gly, Thr, Ser, Ala, Pro, Asp, Glu, Arg und Lys. Besonders bevorzugt wird ein Linker verwendet, in dem 2 - 8 dieser zusätzlichen Aminosäuren die negativ geladenen Aminosäuren Asp und/oder Glu sind.For the purposes of the present invention, 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.
Die Herstellung einer DNA, welche für das Fusionsprotein codiert, kann nach den bekannten Verfahren, wie sie bei Sambrook, J. et al. (1989) (6) beschrieben sind, erfolgen.The preparation of a DNA which codes for the fusion protein can be carried out according to the known methods as described in Sambrook, J. et al. (1989) (6).
Als Streptavidin kann beispielsweise Streptavidin, wie in der EP-B 0 198 015 (7) und EP-A 0 612 325 (8) beschrieben, verwendet werden. Weitere Streptavidin-Derivate oder - Fragmente, wie beispielsweise von Sano, T. et al., (9) beschrieben, sind ebenfalls geeignet. Bevorzugt wird ein Streptavidin verwendet, welches am N-Terminus und/oder C-Terminus trunkiert (verkürzt) ist. Dadurch wird die Aggregation und Proteolyse verhindert (Sano, T. et al., (9)). Vorzugsweise wird ein Streptavidin verwendet, welches mit den Aminosäuren 10 - 20 beginnt und mit den Aminosäuren 130 - 140 endet (Numerierung analog: Argarana, C. E. et al. (1986) (33)). Besonders bevorzugt wird ein Streptavidin der Aminosäuren 16 - 133 oder 13 - 139 verwendet.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.
Die Herstellung der Fusionsproteine erfolgt durch Expression einer DNA (Nukleinsäuresequenz), welche für das Fusionsprotein codiert, in Mikroorganismen, vorzugs¬ weise in Prokaryonten. Damit das Protein in denaturierter, unlöslicher Form ("inclusion bodies") entsteht, sollte der verwendete Expressionsvektor keine Elemente enthalten, die eine Sekretion des Proteins ins Medium vermitteln. Die Herstellung einer für die Expression geeig¬ neten DNA kann vorzugsweise synthetisch erfolgen. Derartige Verfahren sind dem Fachmann
gelaufig und beispielsweise in Beattie, K L und Fowler, R.F (1991) (34), EP-B 0 424 990 (35), Itakura, K et al (1977) (20) beschrieben Die Nukleinsauresequenz der erfindungsge¬ maßen Proteine kann zweckmäßig modifiziert sein Derartige Modifikationen sind beispiels¬ weiseThe fusion proteins are produced by expressing a DNA (nucleic acid sequence) which codes for the fusion protein in microorganisms, preferably in prokaryotes. In order for the protein to be formed in denatured, insoluble form ("inclusion bodies"), 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
— Veränderung der Nukleinsauresequenz, um verschiedene Erkennungssequenzen von Restriktionsenzymen zur Erleichterung der Schritte der Ligation, Klonierung und Mutagenese einzuführen- Change the nucleic acid sequence to introduce various restriction enzyme recognition sequences to facilitate the steps of ligation, cloning and mutagenesis
— Veränderung der Nukleinsauresequenz zum Einbau von bevorzugten Codons für die Wirtszelle- Change the nucleic acid sequence to incorporate preferred codons for the host cell
— Ergänzung der Nukleinsauresequenz um zusatzliche Regulations- und Transkriptionsele¬ mente, um die Expression in der Wirtszelle zu optimieren- Supplementing the nucleic acid sequence with additional regulatory and transcription elements in order to optimize expression in the host cell
Alle weiteren Verfahrensschritte zur Herstellung von geeigneten Expressionsvektoren und zur Expression sind Stand der Technik und dem Fachmann gelaufig Beschrieben sind derartige Methoden beispielsweise bei Sambrook, J et al (1989) fö)All further process steps for the production of suitable expression vectors and for expression are state of the art and are familiar to the person skilled in the art. Such methods are described, for example, by Sambrook, J et al (1989) fö)
Fusionsproteine können sowohl in Prokaryonten als auch in anderen Zellen, beispielsweise in eukaryontischen Wirtszellen, wie Hefen (z B Saccharomyces, Pichia, Hansenula und Kluyveromyces) und Pilzen, wie Aspergillis und Trichoderma in Form unlöslicher Protein¬ aggregate, sogenannten "inclusion bodies" (IBs), anfallen Inclusion bodies entstehen dann, wenn die Synthesegeschwindigkeit des Proteins in der Zelle großer ist als die Faltungsge¬ schwindigkeit zum aktiven nativen Protein In diesem Fall aggregiert das Protein in der Zelle, vorzugsweise im Cytoplasma Dort wird das Protein in denaturierter, verdichteter und unlösli¬ cher Form in der Zelle abgelagert Dadurch erfahrt die Zelle eine möglichst geringe Störung ihrer anderen ZellfünktionenFusion 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
Als prokaryontische Wirtsorganismen sind beispielsweise Escherichia, Streptomyces oder Bacillus geeignet Zur Herstellung der erfindungsgemäßen Fusionsproteine werden die Mikro¬ organismen, vorzugsweise Prokaryonten, in üblicher Weise mit dem Vektor, welcher die für das Fusionsprotein codierende DNA enthalt, transformiert und anschließend in üblicher Weise fermentiert Nach Aufschluß der Zellen wird das unlösliche, inaktive Protein (IBs) in üblicher Weise, beispielsweise durch Zentrifugation (Pelletfraktion) isoliert Die gewünschten unlos-
liehen Proteinaggregate können ggf. durch Waschen des Pellets, mit z. B. detergentienhaltigen Puffern, weiter angereichert werden.Suitable prokaryotic host organisms are, for example, Escherichia, Streptomyces or Bacillus. For the production of the fusion proteins according to the invention, 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. After digestion 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.
Die IBs werden nach den dem Fachmann geläufigen Verfahren mit einem Denaturie- rungsmittel, wie z.B. Guanidinhydrochlorid, Harnstoff oder einem Harnstoffderivat (vgl. z. B. US Patent Nr. 5,453,363) solubilisiert und durch Verdünnung oder Dialyse in einen geeigneten nicht denaturierenden Puffer (pH >8.5) überführt. Die Verdünnung erfolgt dabei in einer solchen Weise, daß das verbleibende Denaturierungsmittel die enzymatische Hydrolyse des Fusionsproteins nicht wesentlich beeinflußt.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. 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.
Vorzugsweise erfolgt die Verdünnung pulsartig, beispielsweise durch Eintropfen des IB- Solubilisats in Puffer (pH >8.5), der kein Denaturierungsmittel enthält.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.
Eine solche pulsartige Verdünnung ermöglicht eine praktisch gleichzeitige Entfernung der Wirkung des Denaturierungsmittels und Vereinzelung der zu solubilisierenden Moleküle. Dadurch wird eine nicht erwünschte intermolekulare Wechselwirkung (Aggregation) der zu solubilisierenden Moleküle weitgehend vermieden.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.
Überraschenderweise hat es sich gezeigt, daß die mit dem erfindungsgemäßen Verfahren hergestellte Fusionsproteine in den Wirtszellen nicht abgebaut werden und sich enzymatisch vollständig spalten lassen, ohne daß im Peptidanteil selbst (z. B. PTH oder Urodilatin) in nennenswertem Umfang eine Spaltung stattfindet.Surprisingly, it has been shown that 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).
Das erfindungsgemäße Verfahren ist insbesondere geeignet zur Herstellung von Urodilatin, Parathormon und deren Fragmente. Besonders bevorzugt werden die Urodilatinfragmente der Aminosäuren 95 - 126, 99 - 126 oder 102 - 126 sowie das Parathormonfragment der Ami¬ nosäuren 1 - 37 hergestellt.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.
Die folgenden Beispiele, Publikationen, die Sequenzprotokolle und die Abbildungen erläutern die Erfindung, deren Schutzumfang sich aus den Patentansprüchen ergibt, weiter. Die beschriebenen Verfahren sind als Beispiele zu verstehen, die auch noch nach Modifikationen den Gegenstand der Erfindung beschreiben.The following examples, publications, the sequence listing and the illustrations further explain the invention, the scope of which is evident from the patent claims. The described methods are to be understood as examples which describe the subject matter of the invention even after modifications.
Fig. 1 zeigt die gemäß Beispiel 1 erhaltenen DNA-Segmente A und B.1 shows the DNA segments A and B obtained according to Example 1.
Fig. 2 zeigt die gemäß Beispiel 2 erhaltenen DNA-Segmente C und D.
Fig. 3 zeigt das gemäß Beispiel 3 erhaltene DNA-Segment E und das gemäß Beispiel2 shows the DNA segments C and D obtained according to Example 2. 3 shows the DNA segment E obtained according to Example 3 and that according to Example
4 erhaltene DNA-Segment F.4 obtained DNA segment F.
Beispiel 1example 1
Konstruktion des core-SA-URO(95-126) Fusionsgens mit EndoproteinaselinkerConstruction of the core-SA-URO (95-126) fusion gene with endoproteinase linker
(Plasmid: pSA-EK-URO)(Plasmid: pSA-EK-URO)
core-SA: verkürztes Streptavidin der Aminosäuren Met-(13 - 139)core-SA: shortened streptavidin of the amino acids Met- (13 - 139)
URO (95 - 126): Urodilatin oder Cardiodilatinfragment der Aminosäuren 95 - 126 (Sequenz beschrieben in Drummer, C. et al. (1993) (24)).URO (95-126): urodilatin or cardiodilatin fragment of amino acids 95-126 (sequence described in Drummer, C. et al. (1993) (24)).
Der Expressionsvektor für das core-SA-URO(95-126) Fusionsgen mit Endoproteinase LysC- Spaltstelle basiert auf dem Expressionsvektor pSAM-CORE für core-Streptavidin. Die Herstellung und Beschreibung des Plasmids pSAM-CORE ist in der WO 93/09144 (11) beschrieben. Zur Konstruktion von core-SA Fusionsproteinen wurde die singuläre am 3'-Ende lokalisierte Nhel Restriktionsschnittstelle vor dem Stopcodon des core-SA Gens benutzt.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.
Ein ca. 140 Bp langes für den Linker [VDDDDK] (SEQ ID NO: l) und das Urodilatin(95- 126) Polypeptid [TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY] (SEQ ID NO: 2) kodierendes DNA-Fragment wurde aus 2 ca. 70 Bp langen chemisch hergestelllten DNA Segmenten zusammengesetzt. Beim "Gendesign" wurden die in E. coli bevorzugt benutzten Codone (E. coli "Codonusage") berücksichtigt und die einzelnen DNA Segmente mit geeig¬ neten singulären Restriktionsendonukleaseschnittstellen an den Enden versehen.An approx. 140 bp long DNA fragment coding for the linker [VDDDDK] (SEQ ID NO: 1) and the urodilatin (95-126) polypeptide [TAPRSLRRSSCFGGRMDRIGAQSGLGCNSFRY] (SEQ ID NO: 2) became 2 approx. 70 bp long chemically produced DNA segments assembled. In the "gene design", the codons used with preference in E. coli (E. coli "Codonusage") were taken into account and the individual DNA segments were provided with suitable singular restriction endonuclease interfaces at the ends.
In zwei Ansätzen wurden die komplementären Oligonukleotide \ (SEQ ID NO: 3) und 2 (SEQ ID NO:4)In two approaches, the complementary oligonucleotides \ (SEQ ID NO: 3) and 2 (SEQ ID NO: 4)
11
AATTCGCTAGCGTTGACGACGATGACAAAACGGCGCCGCGTTCCCTGCGTAGATC TTCCTGCTTCGGC (SEQIDNO:3)
2AATTCGCTAGCGTTGACGACGATGACAAAACGGCGCCGCGTTCCCTGCGTAGATC TTCCTGCTTCGGC (SEQIDNO: 3) 2
GGCCGCCGAAGCAGGAAGATCTACGCAGGGAACGCGGCGCCGTTTTGTCATCGTC GTCAACGCTAGCG (SEQIDNO:4)GGCCGCCGAAGCAGGAAGATCTACGCAGGGAACGCGGCGCCGTTTTGTCATCGTC GTCAACGCTAGCG (SEQIDNO: 4)
zu dem DNA Segment A (Fig. 1) und die Oligonukleotide 3 (SEQ ID NO:5) und 4 (SEQ ID NO:6)to DNA segment A (FIG. 1) and oligonucleotides 3 (SEQ ID NO: 5) and 4 (SEQ ID NO: 6)
33
GGCCGCATGGACCGTATCGGTGCTCAGTCCGGACTGGGTTGCAACTCCTTCCGTT ACTAATGA (SEQ ID NO:5)GGCCGCATGGACCGTATCGGTGCTCAGTCCGGACTGGGTTGCAACTCCTTCCGTT ACTAATGA (SEQ ID NO: 5)
44
AGCTTCATTAGTAACGGAAGGAGTTGCAACCCAGTCCGGACTGAGCACCGATACG GTCCATGC (SEQ ID NO:6)AGCTTCATTAGTAACGGAAGGAGTTGCAACCCAGTCCGGACTGAGCACCGATACG GTCCATGC (SEQ ID NO: 6)
zu dem DNA Segment B, (Fig. 1) "annealt" (Reaktionspuffer: 12,5 mmol/l Tris-HCl, pH 7,0 und 12,5 mmol/l MgCl2 ; Oligonukleotid-Konzentration: jeweils 1 pmol / 60 μl) und die Hybridisierungsprodukte A und B jeweils in die Polylinkerregion des E. coli pUCBM21 Vektors (Boehringer Mannheim GmbH, Mannheim, Deutschland) subkloniert (DNA Segment A, Schnittstellen: EcoRI und Notl; DNA Segment B, Schnittstellen: Notl und Hindill). Mittels DNA Sequenzierung wurde die DNA Sequenz der beiden subklonierten DNA Segmente bestätigt. Danach wurde das Expressionsplasmid pSA-EK-URO für das core-SA- URO(95-126) Fusionsgen in einer Dreifragrnentligation aus dem Nhe/Notl-DNA Segment A , dem Notl/Hindlll-DNA Segment B und dem ca. 2,9 kBp langen Nhel/Hindlll-pSAM-CORE Vektorfragment zusammengesetzt. Dabei wurden die DNA Segmente A und B nach Doppel¬ verdau mit den entsprechenden Endonukleasen aus den entsprechenden pUCBM21 Plasmid- derivaten isoliert. Das gewünschte Plasmid pSA-EK-URO wurde durch Restriktionskartierung identifiziert und die DNA Sequenz des Linker-Urodilatin-Bereichs erneut durch DNA Sequenzierung überprüft.
Beispiel 2to 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 μl 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. Example 2
Konstruktion des core-SA-PTH(l-37) Fusionsgens mit EnterokinaselinkerConstruction of the core-SA-PTH (l-37) fusion gene with enterokinase linker
(Plasmid: pSA-EK-PTH)(Plasmid: pSA-EK-PTH)
PTH (1 - 37): Parathormonfragment der Aminosäuren 1 - 37, Aminosäuresequenz beschrie¬ ben in Handy, G.N. et al., Proc. Natl. Acad. Sei. USA 78 (1981) 7365 - 7369 (39).PTH (1-37): 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).
Der Vektor pSA-EK-PTH zur Expression des core-SA-PTΗ(l-37) Fusionsgens mit Enteroki- nasespaltstelle wurde gemäß der im Beispiel 1 beschriebenen Strategie für das core-SA- URO(95-126) Fusionsgen mit Enterokinasespaltstelle hergestellt.The vector pSA-EK-PTH for expressing the core-SA-PTΗ (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.
In zwei Ansätzen wurden die komplementären Oligonukleotide 5 (SEQ ID NO:7) und 6 (SEQ ID NO.8)In two approaches, the complementary oligonucleotides 5 (SEQ ID NO: 7) and 6 (SEQ ID NO.8)
55
AATTCGCTAGCGGTACCGTCGACGACGATGACAAATCCGTTTCCGAAATCCAGCT GATGCACAACCTGGGTAAACACCTGAACTC (SEQIDNO:7)AATTCGCTAGCGGTACCGTCGACGACGATGACAAATCCGTTTCCGAAATCCAGCT GATGCACAACCTGGGTAAACACCTGAACTC (SEQIDNO: 7)
66
CATGGAGTTCAGGTGTTTACCCAGGTTGTGCATCAGCTGGATTTCGGAAACGGAT TTGTCATCGTCGTCGACGGTACCGCTAGCG (SEQ ID NO:8)CATGGAGTTCAGGTGTTTACCCAGGTTGTGCATCAGCTGGATTTCGGAAACGGAT TTGTCATCGTCGTCGACGGTACCGCTAGCG (SEQ ID NO: 8)
zu dem DNA Segment C (Fig. 2) und die Oligonukleotide 7 (SEQ ID NO:9) und 8 (SEQ ID NO: 10)to DNA segment C (FIG. 2) and oligonucleotides 7 (SEQ ID NO: 9) and 8 (SEQ ID NO: 10)
77
CATGGAACGTGTTGAATGGCTGCGTAAAAAACTGCAGGACGTTCACAACTTCGTT GCTCTGTAATGA (SEQIDNO:9)CATGGAACGTGTTGAATGGCTGCGTAAAAAACTGCAGGACGTTCACAACTTCGTT GCTCTGTAATGA (SEQIDNO: 9)
88th
AGCTTCATTACAGAGCAACGAAGTTGTGAACGTCCTGCAGTTTTTTACGCAGCCA TTCAACACGTTC (SEQ ID NO: 10)
zu dem DNA Segment D (Fig 2) "annealt" (Reaktionspuffer 12,5 mmol/l Tπs-HCl, pH 7,0 und 12,5 mmol/l MgCl2 , Oligonukleotid-Konzentration jeweils 1 nmol / 60 μl) und die Hybridisierungsprodukte C und D jeweils in die Polylinkerregion des E coli pUCBM21 Vektors subkloniert (DNA Segment C, Schnittstellen EcoRI und Ncol, DNA Segment D, Schnittstellen Ncol und Hindlü) Mittels DNA Sequenzierung wurde die DNA Sequenz der beiden subklonierten DNA Segmente bestätigt Danach wurde das Expressionsplasmid pSA- EK-PTΗ für das core-SA-EK-PTH(l-37) Fusionsgen in einer Dreifragmentligation aus dem Nhel/NcoI-DNA Segment C , dem NcoI/HindüI-DNA Segment D und dem ca 2,9 kBp langen Nhel/Hindlll-pSAM-CORE Vektorfragment zusammengesetzt Dabei wurden die DNA Segmente C und D nach Doppelverdau mit den entsprechenden Endonukleasen aus den entsprechenden pUCBM21 Plasmidderivaten isoliert Das gewünschte Plasmid pSA-EK-PTH wurde durch Restriktionskartierung identifiziert und die DNA Sequenz des Enterokinaselin- ker-PTH-Bereichs erneut durch DNA Sequenzierung überprüftAGCTTCATTACAGAGCAACGAAGTTGTGAACGTCCTGCAGTTTTTTACGCAGCCA TTCAACACGTTC (SEQ ID NO: 10) "Annealed" to DNA segment D (FIG. 2) (reaction buffer 12.5 mmol / l Tπs-HCl, pH 7.0 and 12.5 mmol / l MgCl2, oligonucleotide concentration in each case 1 nmol / 60 μl) and the hybridization products C and D were each subcloned into the polylinker region of the E coli pUCBM21 vector (DNA segment C, interfaces EcoRI and Ncol, DNA segment D, interfaces Ncol and Hindlü). The DNA sequence of the two subcloned DNA segments was confirmed by means of DNA sequencing. The expression plasmid pSA was then confirmed - EK-PTΗ 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
Beispiel 3Example 3
Konstruktion des core-SA-PTH(l-37) Fusionsgens mit Thrombinlinker (Plasmid: pSA-Construction of the core-SA-PTH (1-37) fusion gene with thrombin linker (plasmid: pSA-
THRO-PTH)THRO-PTH)
Das Plasmid pSA-THRO-PTH leitet sich von dem core-SA-EK-PTH Expressionsplasmid pSA-EK-PTH (siehe Beispiel 2) ab, indem die kodierende Region für den Enterokinaselinker durch einen Thrombinlinker ersetzt wurdeThe 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
Die Aminosauresequenz des verwendeten Thrombinlinkers [GDFLAEGLVPR] (SEQ ID NO 15) basiert auf der naturlichen Thrombinspaltstelle in Fibrinogen (Aminosaureposition 6- 16) und der minimalen Erkennungssequenz für Thrombin (Carter, P In Ladisch, M R , Willson, R C , Painton, C C , Builder, S E eds (1990) (12))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))
Dazu wurde das Plasmid pSA-EK-PTH mit Nhel und PvuII verdaut, das ca 2,9 kBp lange Nhel/PvuII-pSA-EK-PTH Vektorfragment isoliert und mit dem aus den 2 komplementären Oligonukleotiden 9 (SEQ ID NO 11) und 10 (SEQ ID NO 12) durch Hybridisierung herge¬ stellten DNA Segment E (Fig 3) ligiertFor this purpose, 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
99
CTAGCCCGGGTGACTTCCTGGCTGAAGGTCTGGTTCCGCGTTCCGTTTCCGAAATC CAG (SEQ ID NO 11)
10CTAGCCCGGGTGACTTCCTGGCTGAAGGTCTGGTTCCGCGTTCCGTTTCCGAAATC CAG (SEQ ID NO 11) 10
CTGGATTTCGGAAACGGAACGCGGAACCAGACCTTCAGCCAGGAAGTCACCCGG G (SEQ ID NO: 12)CTGGATTTCGGAAACGGAACGCGGAACCAGACCTTCAGCCAGGAAGTCACCCGG G (SEQ ID NO: 12)
Die gewünschte Plasmidkonstruktion pSA-THRO-PTH wurde durch Restriktionskartierung identifiziert und die ausgetauschte Linkeπegion durch DNA Sequenzierung überprüft.The desired plasmid construction pSA-THRO-PTH was identified by restriction mapping and the exchanged left region was checked by DNA sequencing.
Beispiel 4Example 4
Konstruktion des core-SA-PTH(l-37) Fusionsgens mit TEV Linker (Plasmid: pSA-Construction of the core-SA-PTH (l-37) fusion gene with TEV linker (plasmid: pSA-
TEV-PTH)TEV-PTH)
Das Plasmid pSA-TEV-PTH leitet sich von dem core-SA-EK-PTH Expressionsplasmid pSA- EK-PTH (siehe Beispiel 2) ab, indem die kodierende Region für den Enterokinaselinker durch einen TEV Linker ersetzt wurde.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.
Die Pflanzenvirus TEV NIa Proteinase ("tobacco etch virus") erkennt die Aminosäuresequenz ENLYFQiG/S und spaltet zwischen Gin und Gly oder Ser (Dougherty, W.G. et al., (1988)) (13). Das rekombinat hergestellte Enzym wurde von GIBCO BRL (Life Technologies, Ine Gaithersburg, MD, USA) bezogen.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). The recombinantly produced enzyme was obtained from GIBCO BRL (Life Technologies, Ine Gaithersburg, MD, USA).
Dazu wurde das Plasmid pSA-EK-PTH mit Nhel und PvuII verdaut, das ca. 2,9 kBp lange Nhel/PvuII-pSA-EK-PTH Vektorfragment isoliert und mit dem aus den 2 komplementären Oligonukleotiden JI (SEQ ID NO: 13) und \2 (SEQ ID NO: 14) durch Hybridisierung herge¬ stellten DNA Segment F (Fig. 3) ligiert.For this purpose, 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.
iiii
CTAGCGGATCCGAAAACCTGTACTTCCAGTCCGTTTCCGAAATCCAG (SEQ ID NO: 13)CTAGCGGATCCGAAAACCTGTACTTCCAGTCCGTTTCCGAAATCCAG (SEQ ID NO: 13)
1111
CTGGATTTCGGAAACGGACTGGAAGTACAGGTTTTCGGATCCG (SEQ ID NO: 14)
Die gewünschte Plasmidkonstruktion pSA-TEV-PTH wurde durch Restriktionskartierung identifiziert und die ausgetauschte Linkerregion durch DNA Sequenzierung überprüft.CTGGATTTCGGAAACGGACTGGAAGTACAGGTTTTCGGATCCG (SEQ ID NO: 14) The desired plasmid construction pSA-TEV-PTH was identified by restriction mapping and the exchanged linker region was checked by DNA sequencing.
Beispiel 5Example 5
Expression der core-SA Fusionsproteine in E. coliExpression of core-SA fusion proteins in E. coli
Zur Expression der core-SA Fusionsproteine wurde der E. coli Kl 2 Stamm RM82 (eine Methionin Revertante von ED 8654, Murray, N.E. et al.(1977)) (14) jeweils mit einem der in den Beispielen 1 - 4 beschriebenen Expressionsplasmiden pSA-EK-URO, pSA-EK-PTH, pSA-THRO-PTH und pSA-TEV-PTH (Ampicillin-Resistenz) und dem lacW-Repressorplas- mid pUBS500 (Kanamycin-Resistenz, Herstellung und Beschreibung siehe: EP-A 0368342) transformiert.To express the core-SA fusion proteins, 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.
Die RM82/pUBS500/pSA-EK-URO, RM82/pUBS500/pSA-EK-PTH, RM82/pUBS500/pSA- THRO-PTH und RM82/pUBS500/pSA-TEV-PTH Zellen wurden in DYT-Medium (1% (w/v) Hefeextrakt, 1% (w/v) Bacto Tryptone (Difco, Detroit, USA) und 0,5% NaCl), mit 50 mg/l Ampicillin und 50 mg/l Kanamycin bis zu einer optischen Dichte bei 550 nm von 0,6 - 0,9 angezogen und anschließend mit IPTG (Isopropyl-ß-D-thiogalactosid) (1 - 5 mmol/l Endkon¬ zentration) induziert. Nach einer Induktionsphase von 4 - 8 Stunden wurden die Zellen durch Zentrifugation geerntet und die Zellpellets mit 25 mmol/l Kaliumphosphatpuffer, pH 7,5 gewaschen.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-β-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, pH 7.5.
ExpressionsanalyseExpression analysis
Die Zellpellets aus jeweils 1 ml abzentrifügiertem Anzuchtmedium (RM82/pUBS500/pSA- EK-URO, RM82/pUBS500/pSA-EK-PTH, RM82/pUBS500/pSA-THRO-PTH undThe 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/ρUBS500/pSA-TEV-PTH Zellen) wurden in 0,25 ml 10 mmol/l Phosphatpuffer, pH 6,8 und 1 mmol/l EDTA resuspendiert und die Zellen durch Ultraschallbehandlung aufge¬ schlossen. Nach Zentrifugation wurde der Überstand mit 1/5 Volumen 5xSDS-Probenpuffer (lxSDS-Probenpuffer: 50 mmol/l Tris-HCl, pH 6,8, 1% SDS, 1% Mercaptoethanol, 10% Glycerin, 0.001% Bromphenolblau) versetzt. Die unlösliche Zelltrümmerfraktion wurde in 0,3 ml lxSDS-Probenpuffer mit 6 - 8 M Harnstoff resuspendiert, die Proben 5 Minuten bei 95°C inkubiert und zentrifugiert. Danach wurden die Proteine durch SDS-Polyacrylamid Gelelek¬ trophorese (PAGE) aufgetrennt (Laemmli, U.K. (1970)) (15) und mit Coomassie Brilliant Blue R Farbstoff angefärbt.
Die in E. coli synthetisierten core-SA Fusionsproteine waren homogen und wurden ausschließlichlich in der unlöslichen Zelltrümmerfraktion gefunden (IBs) Die Expressionshohe für die core-SA Fusionsproteine betrug 30 - 50% bezogen auf das E. coli GesamtproteinRM82 / ρUBS500 / 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. 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
Beispiel 6Example 6
Zellyse und Präparation der "inclusion bodies" (IBs)Cell lysis and preparation of the "inclusion bodies" (IBs)
Jeweils 200 g (Naßgewicht) E coli RM82/pUBS500/pSA-EK-URO, RM82/pUBS500/pSA- EK-PTH, RM82/pUBS500/pSA-THRO-PTH und RM82/pUBS500/pSA-TEV-PTH Zellen wurden in 1 1 0, 1 mol/1 Tris-HCl, pH 7,0 bei 0°C suspendiert, 300 mg Lysozym zugegeben und 20 Minuten bei 0°C inkubiert Danach wurden die Zellen mechanisch mittels Hochdruck¬ dispersion vollständig aufgeschlossen und die DNA durch Zugabe von 2 ml 1 mol/1 MgCl2 und 10 mg DNAse (Boehringer Mannheim # 154709) bei 25°C in 30 Minuten verdaut Anschließend wurden zur Aufschlußlosung 500 ml 60 mmol/l EDTA, 6% Triton® XI 00 und 1,5 mol/1 NaCl, pH 7,0 zugemischt und weitere 30 Minuten bei 0°C inkubiert Danach wurden die unlöslichen Bestandteile (Zelltrummer und IBs) durch Zentrifugation sedimentiertEach 200 g (wet weight) 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
Das Pellet wurde in 1 1 0,1 mol/1 Tris-HCl, 20 mmol/l EDTA, pH 6,5 suspendiert, 30 Minuten bei 25°C inkubiert und das IB-Präparat durch Zentrifugation isoliertThe 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
Solubilisierung der IBsSolubilization of IBs
25 g IB-Pellet (Naßgewicht) wurden in 200 ml 10 mmol/l Tris-HCl Puffer, 8 moL/1 Harnstoff, 10 mmol/l EDTA, pH 7,0 durch 2-stundiges Ruhren bei 25°C suspendiert Die unlöslichen Bestandteile wurden durch Zentrifugation abgetrennt und der klare Überstand weiterverarbei¬ tet25 g of IB pellet (wet weight) 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
Beispiel 7Example 7
Verdünnung des SolubilisatsDilution of the solubilizate
Die Verdünnung wurde in einem BioFloII-Fermenter (New Brunswick Scientific Co Ine , Edison, N J , USA) bei 25°C unter Rühren (300 UPM) durch kontinuierliche Zugabe von 200 ml Core-SA-Fusionsprotein-Solubilisat in 3,8 1 50 mmol/l Tris HCl pH 9,0 mittels einer Pumpe (Fordermenge 15-40 ml/Std ) bewirkt
Beispiel 8The dilution was carried out in a BioFloII fermenter (New Brunswick Scientific Co. Ine, Edison, NJ, USA) at 25 ° C. with stirring (300 rpm) by continuously adding 200 ml of Core SA fusion protein solubilisate in 3.8 l of 50 mmol / l Tris HCl pH 9.0 by means of a pump (flow rate 15-40 ml / hour) Example 8
Enzymatische Spaltung der Core-SA FusionsproteineEnzymatic cleavage of the core-SA fusion proteins
Enterokinase Spaltung ( Val Asp4Lys- Spaltsequenz)Enterokinase cleavage (Val Asp4Lys cleavage sequence)
Die core-SA Fusionsproteine mit Enterokinaseschnittstelle wurden in einer Konzentration von 0,3 bis 3 mg/ml und einem Substrat / Proteinase Verhältnis von 1:20 bis 1 :250 (Enterokinase, Restriktionsproteinase aus Kälberdarm, Boehringer Mannheim, Mannheim, Deutschland) in 50 mmol/l Tris-HCl, pH 8,0 bei 30°C verdaut und der zeitliche Verlauf der enzymatischen Spal¬ tung (Kinetik) durch analytische Reversed Phase HPLC (siehe Beispiel 9.1) analysiert. Dazu wurden aus dem Reaktionsansatz über einen Zeitraum von 6 bis 24 Stunden Proben ( 10 bis 100 μl) im Abstand von 1 bis 3 Stunden entnommen.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 μl) were taken at intervals of 1 to 3 hours from the reaction mixture over a period of 6 to 24 hours.
LysC Endoproteinase Spaltung (Lys- Spaltstelle)LysC endoproteinase cleavage (Lys cleavage site)
Das Core-SA-EK-URO Fusionsprotein wurde in einer Konzentration von 0,3 bis 3 mg/ml und einem Substrat / Proteinase Verhältnis von 1:1000 bis 1 :25000 ( LysC Endoproteinase aus Lysobacter enzymogenes, sequencing grade; Boehringer Mannheim, Mannheim, Deutschland) in 50 mmol/l Tris-HCl, pH 8,0 bei 30 bis 35°C verdaut und der zeitliche Verlauf der enzymatischen Spaltung durch analytische Reversed Phase HPLC (siehe Beispiel 9.1) analysiert. Dazu wurden aus dem Reaktionsansatz über einen Zeitraum von 6 bis 24 Stunden Proben (10 bis 100 μl) im Abstand von 1 bis 3 Stunden entnommen.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 μl) were taken at intervals of 1 to 3 hours from the reaction mixture over a period of 6 to 24 hours.
Thrombin Spaltung (GDFLAEGLVPR-Spaltsequenz)Thrombin cleavage (GDFLAEGLVPR cleavage sequence)
Das Core-SA-THRO-PTH Fusionsprotein wurde in einer Konzentration von 0,3 bis 3 mg/ml und einem Substrat / Proteinase Verhältnis von 1 :50 bis 1 :500 (Thrombin aus Humanplasma, Boehringer Mannheim, Mannheim, Deutschland) in 50 mmol/l Tris-HCl, pH 8,8 bei 25 bis 30° C verdaut und der zeitliche Verlauf der enzymatischen Spaltung durch analytische Reversed Phase HPLC (siehe Beispiel 9.1) analysiert. Dazu wurden aus dem Reaktionsansatz über einen Zeitraum von 6 bis 24 Stunden Proben (10 bis 100 μl) im Abstand von 1 bis 3 Stunden entnommen.
TEV NIa Proteinase Spaltung (GluAsnLeuTyrPheGln-i-Gly/Ser-Spaltsequenz)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 μl) 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)
Das Core-SA-TEV-PTH Fusionsprotein wurde in einer Konzentration von 0,3 bis 3 mg/ml und einem Substrat / Proteinase Verhältnis von 1 :50 bis 1:500 (rekombinante TEV NIa Restriktionsproteinase, GIBCO BRL Life Technologies, Inc. Gaithersburg, MD, USA) in 50 mmol/l Tris-HCl, pH 8,0, 0,5 mmol/l EDTA und 1 mmol/l DTT bei 30°C verdaut und der zeitliche Verlauf der enzymatischen Spaltung durch analytische Reversed Phase HPLC (siehe Beispiel 9.1) analysiert. Dazu wurden aus dem Reaktionsansatz über einen Zeitraum von 6 bisThe 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). For this purpose, the reaction mixture was used over a period of 6 to
24 Stunden Proben (10 bis 100 μl) im Abstand von 1 bis 3 Stunden entnommen.24 hours samples (10 to 100 μl) taken at intervals of 1 to 3 hours.
Beispiel 9Example 9
Abtrennung von Core-SA und nicht gespaltenem Core-SA Fusionsprotein durchSeparation of core-SA and uncleaved core-SA fusion protein by
PräzipitationPrecipitation
Das freigesetzte Core-SA Trägerprotein und nicht gespaltenes Core-SA Fusionsprotein wurden aus dem Spaltungsansatz durch pH-Erniedrigung (pH < 6) präzipitiert. Der Spaltungsansatz wurde mit 1 mol/1 Zitronensäure bis zu einer Endkonzentration vonThe 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
25 mmol/l versetzt, auf pH 3,0 eingestellt und das Präzipitat durch Zentrifugation oder Filtra¬ tion abgetrennt.25 mmol / l, adjusted to pH 3.0 and the precipitate separated by centrifugation or filtration.
Beispiel 10Example 10
Reinigung der Peptide URO(95-126) und PTH(l-37)Purification of the peptides URO (95-126) and PTH (l-37)
Die enzymatisch freigesetzten Peptide können mit chromatographischen Methoden, die dem Fachmann bekannt sind, weiter gereinigt werden.The enzymatically released peptides can be further purified using chromatographic methods known to the person skilled in the art.
10.1 Reinigung der Peptide durch Kationenaustauschchromatographie an Fractogel EMD-SO3 650(M)10.1 Purification of the Peptides by Cation Exchange Chromatography on Fractogel EMD-SO3 650 (M)
Der Spaltungsansatz wurde auf eine mit 25 mmol/l Citronensäure, pH 3,0 äquilibrierte Fractogel EMD-SO3"-650(M) Säule (3 x 40 cm, V = 283 ml) der Firma Merck (Darmstadt, Deutschland) beladen (1 SV/Std.) und so lange mit dem Äquilibrierungs- puffer gewaschen, bis die Absorption des Eluats bei 280 nm den Leerwert des Puffers erreichte. Die Elution des gebundenen Materials erfolgte durch einen Gradienten von 0 bis 1 mmol/l NaCl in Äquilibrierungspuffer (10 bis 20 SV, 1 SV/Std.).
10.2 Reinigung der Peptide durch Reversed Phase HPLCThe cleavage mixture was loaded onto a Fractogel EMD-SO3 "-650 (M) column (3 x 40 cm, V = 283 ml) from Merck (Darmstadt, Germany) equilibrated with 25 mmol / l citric acid, pH 3.0 (1st SV / h) and washed with the equilibration buffer until the absorption of the eluate at 280 nm reached the empty value of the buffer. 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
Nach Vorreinigung der Peptide mittels Kationenaustauschchromatographie (siehe: Beispiel 10.1) wurde ein Aliquot von 1 bis 2 ml (ca. 100 bis 300 μg) durch semipräpa- rative RP-HPLC unter Fraktionierung weiter aufgereinigt.After pre-purification of the peptides by means of cation exchange chromatography (see: Example 10.1), an aliquot of 1 to 2 ml (approx. 100 to 300 μg) was further purified by semipreparative RP-HPLC with fractionation.
Chromatographiebedingiingen:Chromatography conditions:
Säule: Eurospher 100-Cg, 5 μm (4 x 250 mm, V = 3,17 ml)Column: Eurospher 100-Cg, 5 μm (4 x 250 mm, V = 3.17 ml)
Knauer, Berlin, Deutschland)Knauer, Berlin, Germany)
Probenvolumen: 1 - 2 ml (100 - 300 μg Protein)Sample volume: 1 - 2 ml (100 - 300 μg protein)
Detektor: UV, 220 nnDetector: UV, 220 nn
Flußrate: 0,5 ml/min Fließmittel:Flow rate: 0.5 ml / min.
A: 0.13% TFA in H2OA: 0.13% TFA in H2O
B: 0.1% TFA, 80% Acetonitril, 20% H2O (v/v)B: 0.1% TFA, 80% acetonitrile, 20% H2O (v / v)
Beispiel 11Example 11
Analytische Reversed Phase (HPLC)Analytical reversed phase (HPLC)
Die analytische reversed phase (HPLC) wurde mit einer Europhersäule durchgeführt (Europher 100-Cg, 5 μm (4 x 250 mm, V = 3, 17 ml, Knauer, Berlin, Deutschland). Das Probenvolumen betrug 10 - 100 μl, entsprechend 1 - 100 μg Protein. Die Detektion erfolgte mit einem UV-Detektor bei 220 nm. Chromatographiert wurde mit einer Flußrate von 0,5 ml/min.The analytical reversed phase (HPLC) was carried out with a Europher column (Europher 100-Cg, 5 μm (4 x 250 mm, V = 3.17 ml, Knauer, Berlin, Germany). The sample volume was 10 - 100 μl, corresponding to 1 100 μg 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.
Fließmittel:Superplasticizer:
A: 0,13 % Trifluoressigsäure in H2O (Gradient 100 - 0 % in 50 min)A: 0.13% trifluoroacetic acid in H 2 O (gradient 100 - 0% in 50 min)
B: 0,1 % Trifluoressigsäure, 80 % Acetonitril, 20 % H2O (v/v) (Gradient 0 - 100 % inB: 0.1% trifluoroacetic acid, 80% acetonitrile, 20% H 2 O (v / v) (gradient 0-100% in
50 min).
Beispiel 1250 min). Example 12
Charakterisierung der gereinigten PeptideCharacterization of the purified peptides
Die Identität und Reinheit der gereinigten Peptide wurde durch Massenspektroskopie (PD-MS und Laser-Desorptionsspektroskopie), analytische reversed phase HPLC, isoelektrische Fokusierung (Bark, J.E. et al., J. Forensic Sei. Soc. 16 (1976) 115 - 120 (42), SDS PAGE (Laemmli, U.K., Nature 227 (1970) 680 - 685 (43)) und Kapillar-Elektrophorese, im Vergleich mit einem chemisch hergestellten Standard, überprüft.
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.
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SEQUENZPROTOKOLLSEQUENCE LOG
(1) ALLGEMEINE ANGABEN:(1. GENERAL INFORMATION:
(i) ANMELDER:(i) APPLICANT:
(A) NAME: BOEHRINGER MANNHEIM GMBH(A) NAME: BOEHRINGER MANNHEIM GMBH
(B) STRASSE: Sandhofer Str. 116(B) STREET: Sandhofer Str. 116
(C) ORT: Mannheim(C) LOCATION: Mannheim
(E) LAND: Germany(E) COUNTRY: Germany
(F) POSTLEITZAHL: D-68305(F) POSTAL NUMBER: D-68305
(G) TELEFON: 08856/60-3446 (H) TELEFAX: 08856/60-3451(G) TELEPHONE: 08856 / 60-3446 (H) TELEFAX: 08856 / 60-3451
(ii) BEZEICHNUNG DER ERFINDUNG: Verfahren zur Herstellung von Peptiden ueber(ii) DESCRIPTION OF THE INVENTION: Process for the preparation of peptides via
Streptavidin-FusionsproteineStreptavidin fusion proteins
(iii) ANZAHL DER SEQUENZEN: 15(iii) NUMBER OF SEQUENCES: 15
(iv) COMPUTER-LESBARE FASSUNG:(iv) COMPUTER READABLE VERSION:
(A) DATENTRÄGER: Floppy disk(A) DISK: Floppy disk
(B) COMPUTER: IBM PC compatible(B) COMPUTER: IBM PC compatible
(C) BETRIEBSSYSTEM: PC-DOS/MS-DOS(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.30B (EPA)(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30B (EPA)
(2) ANGABEN ZU SEQ ID NO: 1:(2) INFORMATION ON SEQ ID NO: 1:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 6 Aminosäuren(A) LENGTH: 6 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 1(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
Val Asp Asp Asp Asp Lys 1 5Val Asp Asp Asp Asp Lys 1 5
(2) ANGABEN ZU SEQ ID NO: 2:(2) INFORMATION ON SEQ ID NO: 2:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 32 Aminosäuren(A) LENGTH: 32 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 2:(ii) MOLECULE TYPE: Peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met 1 5 10 15Thr Ala Pro Arg Ser Leu Arg Arg Ser Ser Cys Phe Gly Gly Arg Met 1 5 10 15
Asp Arg lle Gly Ala Gin Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20 25 30
Asp Arg lle Gly Ala Gin Ser Gly Leu Gly Cys Asn Ser Phe Arg Tyr 20 25 30
(2) ANGABEN ZU SEQ ID NO: 3:(2) INFORMATION ON SEQ ID NO: 3:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 68 Basenpaare(A) LENGTH: 68 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 3: AATTCGCTAG CGTTGACGAC GATGACAAAA CGGCGCCGCG TTCCCTGCGT AGATCTTCCT 60 GCTTCGGC 68(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AATTCGCTAG CGTTGACGAC GATGACAAAA CGGCGCCGCG TTCCCTGCGT AGATCTTCCT 60 GCTTCGGC 68
(2) ANGABEN ZU SEQ ID NO: 4:(2) INFORMATION ON SEQ ID NO: 4:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 68 Basenpaare(A) LENGTH: 68 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 4: GGCCGCCGAA GCAGGAAGAT CTACGCAGGG AACGCGGCGC CGTTTTGTCA TCGTCGTCAA 60 CGCTAGCG 68(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GGCCGCCGAA GCAGGAAGAT CTACGCAGGG AACGCGGCGC CGTTTTGTCA TCGTCGTCAA 60 CGCTAGCG 68
(2) ANGABEN ZU SEQ ID NO: 5:(2) INFORMATION ON SEQ ID NO: 5:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 63 Basenpaare(A) LENGTH: 63 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetische Oligonukleotid"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 5: GGCCGCATGG ACCGTATCGG TGCTCAGTCC GGACTGGGTT GCAACTCCTT CCGTTACTAA 60 TGA 63(A) DESCRIPTION: / desc = "synthetic oligonucleotide" (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GGCCGCATGG ACCGTATCGG TGCTCAGTCC GGACTGGGTT GCAACTCCTT CCGTTACTAA 60 TGA 63
(2) ANGABEN ZU SEQ ID NO: 6:(2) INFORMATION ON SEQ ID NO: 6:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 63 Basenpaare(A) LENGTH: 63 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 6: AGCTTCATTA GTAACGGAAG GAGTTGCAAC CCAGTCCGGA CTGAGCACCG ATACGGTCCA 60 TGC 63(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AGCTTCATTA GTAACGGAAG GAGTTGCAAC CCAGTCCGGA CTGAGCACCG ATACGGTCCA 60 TGC 63
(2) ANGABEN ZU SEQ ID NO: 7:(2) INFORMATION ON SEQ ID NO: 7:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 85 Basenpaare(A) LENGTH: 85 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 7: AATTCGCTAG CGGTACCGTC GACGACGATG ACAAATCCGT TTCCGAAATC CAGCTGATGC 60 ACAACCTGGG TAAACACCTG AACTC 85(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AATTCGCTAG CGGTACCGTC GACGACGATG ACAAATCCGT TTCCGAAATC CAGCTGATGC 60 ACAACCTGGG TAAACACCTG AACTC 85
(2) ANGABEN ZU SEQ ID NO: 8:(2) INFORMATION ON SEQ ID NO: 8:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 85 Basenpaare(A) LENGTH: 85 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear
;ii) ART DES MOLEKÜLS: other nucleic acid(D) TOPOLOGY: linear ; ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 8: CATGGAGTTC AGGTGTTTAC CCAGGTTGTG CATCAGCTGG ATTTCGGAAA CGGATTTGTC 60 ATCGTCGTCG ACGGTACCGC TAGCG 85(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CATGGAGTTC AGGTGTTTAC CCAGGTTGTG CATCAGCTGG ATTTCGGAAA CGGATTTGTC 60 ATCGTCGTCG ACGGTACCGC TAGCG 85
(2) ANGABEN ZU SEQ ID NO: 9:(2) INFORMATION ON SEQ ID NO: 9:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 67 Basenpaare(A) LENGTH: 67 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 9: CATGGAACGT GTTGAATGGC TGCGTAAAAA ACTGCAGGAC GTTCACAACT TCGTTGCTCT 60 GTAATGA 67(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CATGGAACGT GTTGAATGGC TGCGTAAAAA ACTGCAGGAC GTTCACAACT TCGTTGCTCT 60 GTAATGA 67
(2) ANGABEN ZU SEQ ID NO: 10:(2) INFORMATION ON SEQ ID NO: 10:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 59 Basenpaare(A) LENGTH: 59 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 10: CTAGCCCGGG TGACTTCCTG GCTGAAGGTC TGGTTCCGCG TTCCGTTTCC GAAATCCAG 59
(2) ANGABEN ZU SEQ ID NO: 11:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: CTAGCCCGGG TGACTTCCTG GCTGAAGGTC TGGTTCCGCG TTCCGTTTCC GAAATCCAG 59 (2) INFORMATION ON SEQ ID NO: 11:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 59 Basenpaare(A) LENGTH: 59 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 11: CTAGCCCGGG TGACTTCCTG GCTGAAGGTC TGGTTCCGCG TTCCGTTTCC GAAATCCAG 59 (2) ANGABEN ZU SEQ ID NO: 12:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: CTAGCCCGGG TGACTTCCTG GCTGAAGGTC TGGTTCCGCG TTCCGTTTCC GAAATCCAG 59 (2) INFORMATION ON SEQ ID NO: 12:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 55 Basenpaare(A) LENGTH: 55 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 12: CTGGATTTCG GAAACGGAAC GCGGAACCAG ACCTTCAGCC AGGAAGTCAC CCGGG 55 (2) ANGABEN ZU SEQ ID NO: 13:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CTGGATTTCG GAAACGGAAC GCGGAACCAG ACCTTCAGCC AGGAAGTCAC CCGGG 55 (2) INFORMATION ABOUT SEQ ID NO: 13:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 47 Basenpaare(A) LENGTH: 47 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 13: CTAGCGGATC CGAAAACCTG TACTTCCAGT CCGTTTCCGA AATCCAG 47(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: CTAGCGGATC CGAAAACCTG TACTTCCAGT CCGTTTCCGA AATCCAG 47
(2) ANGABEN ZU SEQ ID NO: 14:
(i) SEQUENZKENNZEICHEN:(2) INFORMATION ON SEQ ID NO: 14: (i) SEQUENCE LABEL:
(A) LÄNGE: 43 Basenpaare(A) LENGTH: 43 base pairs
(B) ART: Nucleotid(B) TYPE: nucleotide
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: other nucleic acid(ii) MOLECULE TYPE: other nucleic acid
(A) BESCHREIBUNG: /desc = "synthetisches Oligonukleotid"(A) DESCRIPTION: / desc = "synthetic oligonucleotide"
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 14: CTGGATTTCG GAAACGGACT GGAAGTACAG GTTTTCGGAT CCG 43(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: CTGGATTTCG GAAACGGACT GGAAGTACAG GTTTTCGGAT CCG 43
(2) ANGABEN ZU SEQ ID NO: 15:(2) INFORMATION ON SEQ ID NO: 15:
(i) SEQUENZKENNZEICHEN:(i) SEQUENCE LABEL:
(A) LÄNGE: 11 Aminosäuren(A) LENGTH: 11 amino acids
(B) ART: Aminosäure(B) TYPE: amino acid
(C) STRANGFORM: Einzelstrang(C) STRAND FORM: Single strand
(D) TOPOLOGIE: linear(D) TOPOLOGY: linear
(ii) ART DES MOLEKÜLS: Peptid(ii) MOLECULE TYPE: Peptide
(xi) SEQUENZBESCHREIBUNG: SEQ ID NO: 15:(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:
Gly Asp Phe Leu Ala Glu Gly Leu Val Pro Arg 1 5 10
Gly Asp Phe Leu Ala Glu Gly Leu Val Pro Arg 1 5 10
Claims
1 Verfahren zur rekombinanten Herstellung eines Peptids durch Expression einer DNA in Mikroorganismen, welche für ein Fusionsprotein aus Streptavidin und dem genannten Peptid codiert, wobei Streptavidin und Peptid über eine Peptidsequenz, welche durch eine Endoproteinase spaltbar ist, verbunden sind, Isolierung des unlöslichen, inaktiven Fusionsproteins, Solubilisierung des inaktiven Fusionsproteins mit einem Denaturie¬ rungsmittel, Verdünnung des Denaturierungsmittels bei einem pH- Wert zwischen 8,5 und 11, bis die Spaltung des Fusionsproteins durch eine Endoproteinase durchführbar ist, Spaltung des Fusionsproteins, Erniedrigung des pH-Werts, bis das abgespaltene Streptavidin und nicht gespaltenes Fusionsprotein prazipitieren, und Reinigung des gewünschten Peptids aus dem Überstand.1 Process for the recombinant production of a peptide by expression of a DNA in microorganisms which codes for a fusion protein of streptavidin and the said peptide, streptavidin and peptide being linked via a peptide sequence which is cleavable 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 between 8.5 and 11 until the fusion protein can be cleaved by an endoproteinase, cleavage of the fusion protein, lowering of the pH until the cleaved off Precipitate streptavidin and uncleaved fusion protein, and purify the desired peptide from the supernatant.
2. Verfahren nach Anspruch 1, dadurch gekennzeichnet, daß die Verdünnung des solubili- sierten, inaktiven Proteins in wäßrige Pufferlösung erfolgt.2. The method according to claim 1, characterized in that the dilution of the solubilized, inactive protein is carried out in aqueous buffer solution.
3. Verfahren nach den Ansprüchen 1 - 3, dadurch gekennzeichnet, daß als Peptid Urodilatin, Parathormon oder ein davon abgeleitetes Fragment verwendet wird.3. Process according to claims 1-3, characterized in that urodilatin, parathyroid hormone or a fragment derived therefrom is used as the peptide.
4. Verfahren nach den Ansprüchen 1 - 3, dadurch gekennzeichnet, daß als Peptid ein Fragment von Urodilatin (95 - 126) der Aminosauren 99 - 126 oder 102 - 126 oder ein Fragment von Parathormon der Aminosauren 1 - 37 verwendet wird 4. The method according to claims 1-3, characterized in that a fragment of urodilatin (95-126) of amino acids 99-126 or 102-126 or a fragment of parathyroid hormone of amino acids 1-37 is used as the peptide
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
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DE19542702 | 1995-11-16 | ||
DE19542702 | 1995-11-16 | ||
PCT/EP1996/004850 WO1997018314A1 (en) | 1995-11-16 | 1996-11-06 | Process for the preparation of peptides by way of streptavidin fusion proteins |
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EP0861325A1 true EP0861325A1 (en) | 1998-09-02 |
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EP96938115A Withdrawn EP0861325A1 (en) | 1995-11-16 | 1996-11-06 | Process for the preparation of peptides by way of streptavidin fusion proteins |
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US (1) | US6136564A (en) |
EP (1) | EP0861325A1 (en) |
JP (1) | JP2000500019A (en) |
AU (1) | AU7566396A (en) |
CA (1) | CA2237296C (en) |
WO (1) | WO1997018314A1 (en) |
Families Citing this family (36)
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US20020168718A1 (en) * | 1997-04-03 | 2002-11-14 | California Institute Of Technology | Enzyme-mediated modification of fibrin for tissue engineering |
US7601685B2 (en) * | 1998-08-27 | 2009-10-13 | Eidgenossische Technische Hochschule Zurich | Growth factor modified protein matrices for tissue engineering |
EP2311895B1 (en) | 1999-02-01 | 2017-04-12 | Eidgenössische Technische Hochschule Zürich | Biomaterials formed by nucleophilic addition reaction to conjugated unsaturated groups |
US6958212B1 (en) * | 1999-02-01 | 2005-10-25 | Eidgenossische Technische Hochschule Zurich | Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds |
US7144991B2 (en) | 1999-06-07 | 2006-12-05 | Aletheon Pharmaceuticals, Inc. | Streptavidin expressed gene fusions and methods of use thereof |
EP1190061A1 (en) * | 1999-06-07 | 2002-03-27 | Neorx Corporation | Streptavidin expressed gene fusions and methods of use thereof |
US7291673B2 (en) * | 2000-06-02 | 2007-11-06 | Eidgenossiche Technische Hochschule Zurich | Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds |
EP1465989B1 (en) * | 2001-12-18 | 2008-02-20 | Eidgenossisch Technische Hochschule Zurich | Growth factor modified protein matrices for tissue engineering |
US7247609B2 (en) * | 2001-12-18 | 2007-07-24 | Universitat Zurich | Growth factor modified protein matrices for tissue engineering |
WO2003052091A1 (en) * | 2001-12-18 | 2003-06-26 | Eidgenossisch Technische Hochschule Zurich | Growth factor modified protein matrices for tissue engineering |
US8282912B2 (en) * | 2002-03-22 | 2012-10-09 | Kuros Biosurgery, AG | Compositions for tissue augmentation |
US8603779B2 (en) | 2004-12-01 | 2013-12-10 | Syntaxin, Ltd. | Non-cytotoxic protein conjugates |
US8512984B2 (en) | 2004-12-01 | 2013-08-20 | Syntaxin, Ltd. | Non-cytotoxic protein conjugates |
US8778634B2 (en) | 2004-12-01 | 2014-07-15 | Syntaxin, Ltd. | Non-cytotoxic protein conjugates |
GB0426394D0 (en) * | 2004-12-01 | 2005-01-05 | Health Prot Agency | Fusion proteins |
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WO2006067221A2 (en) * | 2004-12-22 | 2006-06-29 | Kuros Biosurgery Ag | Michael-type addition reaction functionalised peg hydrogels with factor xiiia incorporated biofactors |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1986002077A1 (en) * | 1984-10-02 | 1986-04-10 | Meade Harry M | Production of streptavidin-like polypeptides |
GB8518753D0 (en) * | 1985-07-24 | 1985-08-29 | Glaxo Group Ltd | Microbiological products |
DE3878231T2 (en) * | 1987-03-02 | 1993-05-27 | Bissendorf Peptide Gmbh | NEW CADIODILATIN FRAGMENT, PROCESS FOR THEIR PRODUCTION AND USE. |
GB8723661D0 (en) * | 1987-10-08 | 1987-11-11 | British Bio Technology | Synthetic gene |
DE3935738A1 (en) * | 1989-10-27 | 1991-05-08 | Forssmann Wolf Georg | DRUGS CONTAINING THE HUMAN PARATHORMONE FRAGMENT (1-37) AS AN ACTIVE AGENT |
ATE253639T1 (en) * | 1991-08-19 | 2003-11-15 | Daiichi Suntory Pharma Co Ltd | METHOD FOR PRODUCING PEPTIDES |
-
1996
- 1996-11-06 WO PCT/EP1996/004850 patent/WO1997018314A1/en not_active Application Discontinuation
- 1996-11-06 JP JP9518551A patent/JP2000500019A/en not_active Ceased
- 1996-11-06 CA CA002237296A patent/CA2237296C/en not_active Expired - Fee Related
- 1996-11-06 EP EP96938115A patent/EP0861325A1/en not_active Withdrawn
- 1996-11-06 AU AU75663/96A patent/AU7566396A/en not_active Abandoned
- 1996-11-06 US US09/068,738 patent/US6136564A/en not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
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See references of WO9718314A1 * |
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CA2237296A1 (en) | 1997-05-22 |
CA2237296C (en) | 2008-10-07 |
US6136564A (en) | 2000-10-24 |
WO1997018314A1 (en) | 1997-05-22 |
AU7566396A (en) | 1997-06-05 |
JP2000500019A (en) | 2000-01-11 |
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