EP0393039B1 - Process for producing peptides by specific cleavage of fusion proteins with collagenases obtained by genetic engineering - Google Patents

Process for producing peptides by specific cleavage of fusion proteins with collagenases obtained by genetic engineering

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Publication number
EP0393039B1
EP0393039B1 EP88907257A EP88907257A EP0393039B1 EP 0393039 B1 EP0393039 B1 EP 0393039B1 EP 88907257 A EP88907257 A EP 88907257A EP 88907257 A EP88907257 A EP 88907257A EP 0393039 B1 EP0393039 B1 EP 0393039B1
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Prior art keywords
pro
gly
sequence
amino acid
protein
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EP88907257A
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German (de)
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EP0393039A1 (en
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Harald Scheidecker
Joachim Daum
Peter Donner
Gerhard Siewert
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Bayer Pharma AG
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Schering AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • C07K14/695—Corticotropin [ACTH]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the genetic engineering processes for the production of polypeptides are based on the transfer of a suitable deoxyribonucleic acid (DNA), which contains codons for the amino acid sequence to be produced, to suitable host cells in a form which enables the replication and expression of the newly introduced genetic information.
  • DNA deoxyribonucleic acid
  • Bacteria such as Escherichia coli are preferably used as host cells for small to medium-sized peptides (less than approx. 100 amino acids).
  • polypeptides of the size range mentioned are very easily attacked by intracellular bacterial proteases, since they are not protected by a pronounced tertiary structure. This can lead to a sharp reduction in yield or even to complete loss of the primary biosynthesis product if the expression is directed in such a way that the polypeptide to be produced is formed directly in the final form or, if need be, with an additional N-terminal methionine residue (so-called direct expression) .
  • direct expression N-terminal methionine residue
  • the desired amino acid sequences can generally be obtained in good yield if they are first synthesized as much larger precursor molecules, so-called fusion proteins.
  • a process variant is therefore preferred, in which a hybrid gene is formed on a plasmid vector by an in vitro combination of a bacterial gene which can be easily expressed and, if possible, also regulated, and the DNA sequence for the peptide to be produced.
  • the hybrid gene contains the original bacterial regulatory elements (promoter, ribosomal binding site and terminator) and as large a part of the codons as possible for the N-terminal amino acids of the bacterial gene product, followed by the codons for the new peptide and a stop codon, after transfer of the plasmid vector into a suitable bacterial host cell by transformation, the hybrid gene controls the biosynthesis of a fusion protein, the N-terminal part of which is derived from the corresponding bacterial protein, during the C-terminal part consists of the amino acid sequence of the peptide to be produced.
  • This fusion protein can be easily produced and isolated by fermentation of the transformed cells in large quantities.
  • hybrid genes Various bacterial genes have been used for the construction of hybrid genes, but preferably those for the enzymes ⁇ -galactosidase, ⁇ -lactamase, anthranilate synthetase, alkaline phosphatase and chloramphenicol acetyltransferase.
  • the last step in the preparation of a peptide according to the procedure outlined above is the exact cleavage of the fusion protein.
  • the hybrid gene must be constructed in such a way that an easily and selectively cleavable peptide bond is present in the later fusion protein between the bacterial part and the new peptide sequence to be produced.
  • the development of a specific and broadly applicable process for the cleavage of fusion proteins has proven to be the biggest and so far not completely satisfactorily solved problem in the genetic engineering production of small and medium-sized peptides.
  • the present invention relates to a specific and therefore generally applicable method for splitting fu tion proteins, which provides the exact amino acid sequence of the desired peptide without leaving unwanted additional amino acids at the N-terminal end.
  • the first genetically engineered peptides such as Somatostatin (K. Itakura et al., Science 198, 1056-1063 [1977]) or the A and B chains of human insulin (DV Goeddel et al., Proc. Natl. Acad. Sei, USA 76, 106-110 [1979]), had been released by cleavage with cyanogen bromide from the corresponding fusion proteins which were derived from ⁇ -galactosidase. Bromocyan selectively cleaves the bond between methionine and the next carboxyl amino acid (Met-X, where X can be any of the 20 amino acids specified by the genetic code). Naturally, this method can only be used for peptides which themselves do not contain methionine; this applies to the three peptides mentioned above.
  • fusion proteins which contain the amino acids lysine, arginine or glutamic acid as a link between the N-terminal and the C-terminal part.
  • the enzymes were trypsin (J. Shine et al., Nature 285, 456-461 [1980]) and clostripain (AD Bennett et al., EPA 0131363), both of which open the bond between Lys-X and Arg-X and endoproteinase Glu-C (AD Bennett et al., EPA 0131363), which cleaves the Glu-X bond.
  • Factor Xa a plasma protease whose normal function is the conversion of prothrombin to thrombin, has been used by K. Nagai and HC Thtfgersen (Nature 309, 810-812 [1984]) to release human ⁇ -globin from a fusion protein Its N-terminal part consisted of 31 amino acids of the ⁇ cII protein. Similarly, T. Imai et al. (J. Biochem. 100, 425-432 [1986]) used the enzyme to produce human prorenin via a fusion protein with 9 additional N-terminal amino acids.
  • enterokinase which cleaves the bond between Lys and X in the sequence (Asp) 4 -Lys-X.
  • RM Belagaje et al. (DNA 3, 120 [1984]) have released human growth hormone with this enzyme from a fusion protein in which this sequence is not far from the N-terminal end, similar to that in Trypsinogen, the natural substrate of enterokinase.
  • EP 20290 describes a method which uses the high specificity of collagenases to cleave fusion proteins of corresponding structure.
  • Collagenases recognize the sequence Pro-Y-Gly-Pro, where Y can be any of the 20 amino acids of the genetic code, and split between Y and Gly.
  • a fusion protein which contains this sequence releases a peptide which at the N-terminal end also contains the amino acids Gly and Pro derived from the collagenase sequence. These can be removed as a dipeptide with another enzyme, the postproline dipeptidyl aminopeptidase (PPDA).
  • PPDA postproline dipeptidyl aminopeptidase
  • German patent application (file number P 3731 875.6) describes a process for the production of adrenocorticotropic hormone via fusion proteins which are derived from the alkaline phosphatase from E. coli.
  • fusion proteins with a tetrapeptide sequence Pro-Y-Gly-Pro between bacterial protein sequence and target peptide, where Y is each of the 20 amino acids of the genetic code, can be cleaved specifically at the predetermined position with clostridiopeptidase A, however the response speed was very slow.
  • J. Germino and D. Bastia have produced a fusion protein which, as the link between the N- and C-terminal region, has a sequence of approx. 60 amino acids from Pro ⁇ -2 collagen contained.
  • The- This protein could be cleaved with collagenases as well as the corresponding collagen itself. However, it is not known at which locations within the sequence of approximately 60 amino acids the fusion protein is cleaved exactly. It was also found that a more or less large residue of this sequence is still present at the N-terminal end of the C-terminal cleavage product. This process variant is therefore unsuitable for the production of polypeptides with a precisely specified N-terminal end.
  • the present invention consists in a process for the preparation of target peptides, which is characterized in that a fusion protein of the formula I,
  • X and Y represent each of the 20 amino acids defined by the genetic code
  • Z 1 represents a bacterial amino acid sequence
  • Z 2 denotes the target peptide from any amino acids of the genetic code
  • the invention also relates to fusion proteins of the formula I and processes for their preparation according to claims 1-6, and to gene structures which code for fusion proteins of the formula I and plasmids or comparable vectors (for example phage vectors) which contain codons for repetitive collagenase interfaces and thus for the production of Fusion proteins of formula I are suitable.
  • alkaline phosphatase alkaline phosphatase, anthranilate synthetase, ⁇ -lactamase, ⁇ -galactosidase, chloramphenicol acetyl transferase etc. come into question.
  • X and Y can be any of the 20 amino acids specified in the genetic code, n preferably means 2 to 10.
  • the advantage of the new process for cleaving fusion proteins over the process from EP 20290 is that the rate of conversion can be increased by orders of magnitude, for example with clostridiopeptidase.
  • Fusion proteins that contain the recognition sequence for collagenase in repetitive form are incomparably easier to cleave than the corresponding fusion proteins with a simple cleavage site for collagenases.
  • examples 1-3 describe the preparation and enzymatic cleavage of 3 model proteins which contain different sequences which can be cleaved by collagenases. The amino acid sequences of these proteins are summarized in Table 1.
  • prephosphatases from E.coli K 12, an intermediate product in the biosynthesis of alkaline phosphatase, which consists of 471 amino acids.
  • the pre-phosphatase is converted into the active enzyme by cleaving off an N-terminal signal sequence of 22 amino acids.
  • the signal sequence of 22 amino acids is not split off to any appreciable extent in the modified phosphatases.
  • alkaline phosphatase RA Bradshaw et al., Proc. Natl. Acad. Sci. USA 81, 4692-4696 [1981]
  • phoA nucleotide sequence of the corresponding gene from E. coli K12
  • Vectors which can be used for the construction of fusion proteins based on alkaline phosphatase have also been described (W. Boidol et al., Mol. Gen. Genet. 185, 510512 [1982]; W.
  • oligonucleotides were inserted in two positions of the phoA gene on pSB94, namely in the NcoI or in the Sphl sequence, which form codons in the modified genes for the amino acid sequences recognizable by collagenases. Restriction maps of pSB94 and the plasmids pSA302, pSA506 and pHS4133 derived therefrom are shown in FIGS. 1-4.
  • the reaction with PPDA according to the scheme already given can only lead to the authentic peptide with the exact amino acid sequence at the N-terminal end if the Y-Gly bond is also cleaved within the repetitive cleavage site closest to the N-terminal end of the peptide.
  • Analytical investigations by gel electrophoresis and HPLC showed no heterogeneity of the cleavage products from fusion proteins with repetitive collagenase interfaces. This indicates that all Y-Gly bonds of the repetitive sequence are cut in the reaction with clostridiopeptidase A.
  • the method according to the invention can of course also be carried out in one step, i.e. with simultaneous implementation with collagenase and PPDA.
  • Plasmid pBR 322 (ATCC 40015). On July 16, 1979 the following was deposited with the DSM, Göttingen:
  • pSA302, pSA506 and pHS4133 The biosynthesis of the three proteins is determined in appropriately transformed bacterial cells by the newly combined plasmids pSA302, pSA506 and pHS4133. These plasmids originated from the vector plasmid pSB94. pSB94 was derived from the plasmid pSB53 (deposited on July 16, 1979 under the number ATCC 40020) according to a described method (W. Boidol, dissertation, Technical University Berlin [1984], see also US Pat. No. 4,375,514).
  • the plasmid pSB94 was prepared as follows:
  • reaction mixture consisting of 60 ⁇ l pSB53 (205 ⁇ g / ml), 60 ⁇ l reaction buffer (20mM Tris HCl, 20mM MgCl 150mM NaCl, 20mM mercaptoethanol, pH 7.5), 18 ⁇ l bovine serum albumin (1mg / ml), 5 ⁇ l Sal I, 5 ⁇ l Xho I and 12 ⁇ l H 2 O, the pSB53 DNA was incubated at 37 ° C for 3 hours.
  • restriction enucleases Sal I and Xho I were obtained from Biolabs (USA).
  • the 6454 bp fragment was isolated from a 0.7% agarose gel by electroelution [T. Maniatis, E. Fritsch, J. Sambrock; Molecular Cloning, 164-165, Cold Spring Harbor Lab. New York (1982)] and linked in a ligase approach via the complementary 5'-overhanging single strand ends to form ring molecules.
  • the ligase reaction with T4 ligase (Biolabs, USA) was carried out in a reaction buffer with 50mM TrisHCl, 10mM MgCl 2 , 20mM dithiothreitol, 50 ⁇ g / ml bovine serum albumin, 1mM ATP, pH 7.8.
  • the ligase mixture (100 ⁇ l volume) was incubated at 13 ° C. for 16 hours.
  • the reaction was stopped by phenol extraction and the DNA was precipitated from the aqueous phase after three ether extractions with ethanol. After centrifugation, the DNA precipitate was dissolved in 50 ⁇ l DNA buffer (45mM TnsHCl, 0.1mM EDTA, pH 7.9) and used to transform competent cells of the Escherichia coli SB44 strain [200 ⁇ l competent cells + 25 ⁇ l DNA from the ligase batch].
  • Plasmid DNA was isolated from the transformants obtained (ampicillin-resistant cells) and the structure of the plasmid was confirmed by restriction analysis.
  • Fig. 1 shows a restriction map of pSB94. (The distances between the individual restriction sequences are given in base pairs (bp), the total size is 6454 bp.)
  • the plasmid carries the gene for ⁇ -lactamase (ampicillin resistance) from pBR322 and the gene for alkaline phosphatase (phoA) from E. coli K12.
  • oligonucleotides were inserted into the recognition sequences for the restriction endonucleases Ncol or Spl, which are located within the phoA gene, by DNA cloning.
  • pHS4133 was then prepared from pSA506 by removing a 27 bp PstI fragment.
  • Fig. 2-4 show restriction maps of the three recombined plasmids:
  • Fig. 2 The numbers 297 and 298 above the partial amino acid sequence indicate the amino acids of the pre-phosphatase, between which the additional 6 amino acids are inserted in the protein SA302 / 1.
  • Fig. 4 The numbers 447 and 448 above the partial amino acid sequence identify the amino acids of the pre-phosphatase, between which the additional 6 amino acids are inserted in the protein HS4133 / 1.
  • Oligonucleotides were analyzed using an automated model 381A DNA synthesizer from 'Applied Biosystems, 850 Lincoln Center Drive, Foster City, Calif. 94 404, USA ', synthesized according to the manufacturer's instructions and purified by gel electrophoresis.
  • DNA was sequenced using the dideoxy method (F. Sanger et al., Proc.Natl. Acad. Sci. USA 74, 5463-5467 [1977]) using the single-strand phage vectors M13mp18 and M13mp19 (C. Yanisch- Perron et al., Gene 33, 103-119 [1985]), which are available from Boehringer (Mannheim), Pharmacia-LKB GmbH and many other suppliers.
  • dideoxy method F. Sanger et al., Proc.Natl. Acad. Sci. USA 74, 5463-5467 [1977]
  • M13mp18 and M13mp19 C. Yanisch- Perron et al., Gene 33, 103-119 [1985]
  • Deoxyadenosine 5- [ ⁇ - 35 S] thiotriphosphate was used for the radioactive labeling (MD Biggin et al., Proc. Natl. Acad.
  • restriction endonucleases EcoRI, PstI, SphI and Haelll as well as DNA ligase (T4) were obtained from Boehringer, Mannheim. Ncol was from New England Biolabs GmbH, 6231 Schwalbach, and polynucleotide kinase (T4) from Pharmacia / PL, 7800 Freiburg i.Br.
  • the plasmid pSB94 was linearized with the restriction endonuclease Ncol.
  • a reaction mixture which contained 14 ⁇ g plasmid DNA and 17 units Ncol in 50 ⁇ l reaction buffer (50 mM Tris HCl, 10 mM MgCl 2 , 100 mM NaCl 2 , 1 mM dithiothreitol, pH 7.5) was kept at 37 ° C. for 7 hours incubated. After extraction of the reaction mixture with phenol, the DNA was precipitated with ethanol, dried and dissolved in the TE buffer (45 mM Tris HCl, 1 mM EDTA, pH 7.5).
  • 5'-CAT GGC CCT GCA GGA CCC-3 'and 5'-C ATG GGG TCC TGC AGG GC-3', which were not phosphorylated on the 5'-OH group, were mixed in aqueous solution and 15 min at 37 ° C pre-incubated. They were then treated with 3.5 ⁇ g linearized pSB94 and 4 units of DNA ligasse (T4) in 50 ⁇ l ligase reaction buffer (20 mM Tris HCl, 10 mM MgCl 2 , 10 mM dithioerythritol, 0.6 mM ATP, pH 7.6) for 2.5 h 20 ° C implemented.
  • the DNA was processed by phenol extraction and fithanol precipitation as described above and used to transform the strain E. coli SB 44.
  • the competent cells were produced and transformed using a method by M. Dagert and SD Ehrlich (Gene 6, 23-28 [1979]).
  • the transformed cells were plated out into single colonies. Colonies whose plasmid DNA contains the above-mentioned oligonucleotide sequences were identified by colony hybridization. Colony hybridization was carried out according to the method described by T. Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA [1982]) given basic protocols in a JP Gergen et al. (Nucleic Acids Research 7, 2115-2136 [1979]).
  • oligonucleotides which had previously been labeled with 5'-P 32 phosphate by reaction with ⁇ P 32 -ATP and polynucleotide kinase (T4), served as the radioactive hybridization reagent.
  • the orientation of the synthetic Ncol fragment of 18 bp was determined by restriction analysis.
  • plasmid DNA was isolated according to standard methods and reacted with the restriction endonuclease HaeIII.
  • the resulting fragment mixtures were analyzed by gel electrophoresis (molecular weight determination by comparison with HaeIII-cut pBR322 in an 8% polyacrylamide gel).
  • the Ncol interface used for the integration of the synthetic fragment of 18 bp is located in pSB94 on a Haelll fragment of 385 bp.
  • This fragment must be missing in the recombined plasmids and replaced by two new fragments, since the 18 bp fragment itself carries a HaeIII sequence.
  • the size of the two new fragments depends on the orientation of the 18 bp fragment: in the desired orientation shown in FIG. 2, fragments of 342 bp and 61 bp are expected, while the opposite orientation fragments of 330 bp and 73 bp.
  • the pSA302 shown in Fig. 2 is one of the plasmids with the desired orientation.
  • the recombined plasmids obtained in this way have an intact Sphl sequence only on one side of the DNA section formed from the two 45mer oligonucleotides. In the plasmids sought, this is at a distance of 257 bp from the closest EcoRI sequence (see FIG. 3), while this distance in the plasmids of the opposite orientation is only 212 bp, just as in pSB94.
  • a reaction mixture of 3 ⁇ g DNA of the plasmid pSA506 and 5 units of the restriction enzyme PstI in 50 ⁇ l reaction buffer (as in 1.1.) was incubated for 2 h at 37 ° C. and as in Example 1.1. worked up.
  • 3 DNA fragments (27 bp, 2420 bp and 4007 bp) were obtained, which were separated from one another by electrophoresis in a 1% agarose gel.
  • the two larger fragments were isolated from the gel by electroelution, combined and with DNA ligase (T4) under the reaction conditions as in Example 1.1. implemented, processed and for transformation used by E.coli SB 44.
  • Plasmid DNA was isolated from some ampicillin-resistant clones.
  • pHS4133 the restriction map of which is shown in Fig. 4.
  • pHS4133 is derived from pSA506 by a deletion which leads to a reduction in the size of the EcoRI / Sphl fragment from 257 to 230 bp. This fragment was as in Example 1.2. cloned and sequenced in M13mp18. The nucleotide sequence shown in Fig. 4 was confirmed.
  • the three proteins were obtained by fermentation of the strains described in part 1 of the examples.
  • E.coli SB44 (pSA506) and E.coli SB44 (pHS4133) obtained. Their biosynthesis is regulated in the same way as that of alkaline phosphatase: it is repressed by inorganic phosphate and, conversely, is induced in low-phosphate media (A. Torriani, Biochem. Biophys. Acta 38, 460-469 [1960]).
  • the second antibody was a goat antiserum against rabbit IgG, to which alkaline phosphatase was coupled as a marker enzyme (Cooper Biomedical Inc., Malvern, USA). 5-bromo-4-chloro-3-indolyl phosphate was used as a color reagent.
  • the three proteins as well as the three above Strains from which they are produced are so similar to one another that the same fermentation and isolation process can be used for all of them.
  • the insoluble cell components were separated by centrifugation (20 min. At 10,000 ⁇ g and 4 ° C.), washed twice with digestion buffer and dissolved in 50 mM Tris HCl, pH 8.0 / 8 M urea (60 ml). The solution was freed from insoluble constituents by centrifugation and kept in ice until further purification by gradient chromatography.
  • Main protein component (approx. 50-70%) of the 2.2.
  • the protein extract obtained in urea buffer is the respective phosphatase derivative.
  • SDS polyacrylamide gels under standard conditions UK Laemmli, Nature 227, 680-685 [1970]
  • the corresponding protein bands were identified using the following criteria: a) The strongest band of the protein mixture has a molecular weight which corresponds to the amino acid sequence given in Table 1. b) This protein can be repressed by phosphate, ie the band is missing if the cells have been fermented as in 2.1., but at 10 times the phosphate concentration.
  • the band is plasmid-specific, it is absent in cells of the plasmid-free strain E. coli SB 44, which under the same conditions as in 2.1. was produced. d) The band reacts specifically in the immunoblot with antiserum against purified alkaline phosphatase from E. coli K12.
  • the absorbance of the eluate at 280 nm was recorded with a flow plotometer.
  • the protein composition of the individual fractions was analyzed by SDS-polyacrylamide gel electrophoresis.
  • the phosphatase derivatives eluted at 50-70 mM NaCl.
  • the appropriate fractions were combined and dialyzed against 400 times the volume of buffer (50 mM Tris HCl, 150 mM NaCl, pH 8.0).
  • the proteins remained dissolved under these conditions and were used in this form for the subsequent cleavage with collagenase.
  • the purity of the preparations was checked by SDS-polyacrylamide gel electrophoresis (see p. 35, lanes 4, 6 and 8), it was greater than 90%.
  • the protein content was determined according to W.
  • Clostridiopeptidase A from Clostridium histolyticum (EC 3.4.24.3) was obtained from Sigma Chemie GmbH, D-8024 Deisenhofen, Grünwalder Weg 30 (Type VII, order no. C0773). The enzyme was purified by gradient chromatography using the FPLC and the same anion exchange column as in Example 2.4. further cleaned.
  • collagenase activity was determined using one of E. Wünsch et al. (Hoppe-Seyler's Z. Physiol. Chem. 333, 149-151 [1963]).
  • the most active fraction had a protein content of 0.8 mg / ml and a specific activity of 2500 units / mg.
  • the cleavage tests described below were carried out with this fraction, which was kept at -20 ° C. 3.2. Cleavage of HS 4133/1
  • the single-chain protein has a length of 477 amino acids (molecular weight 49,919). It contains the sequence that can be cleaved by collagenases
  • reaction mixture which contained 53 ⁇ g HS 4133/1 and 0.1 unit clostridiopeptidase A in 100 ⁇ l reaction buffer (90 mM Tris HCl, 5 mM CaCl 2 , 120 mM NaCl, pH 8.0) was incubated at 37 ° C. for 2.5 h. Samples of 10 ⁇ l each were taken at intervals of 15 to 30 minutes and stopped immediately by adding 3 ⁇ l of sample buffer (10% sucrose, 10% sodium dodeeyl sulfate, 1 mM dithiothreitol, 2 mM EDTA) and heating to 95 ° C. for five minutes.
  • sample buffer 10% sucrose, 10% sodium dodeeyl sulfate, 1 mM dithiothreitol, 2 mM EDTA
  • the samples were separated by electrophoresis in an SDS-polyacrylamide gradient gel (gradient from 12.5% to 25% acrylamide with a constant 0.33% bisacrylamide). After staining with Coomassie Brilliant Blue G, the gel became the protein strips cut into strips and evaluated quantitatively at 280 nm using a gel scanner (Isco, optical unit type 6, absorption monitor UA-5). With the help of a corresponding calibration curve, which had been obtained with a purified phosphatase protein, the protein contents for the bands of the largest cleavage product were determined from the peak heights. The results are summarized on p. .36. The product results in a product formation rate of 21 .0 pmol / min / enzyme unit for HS 4133/1.
  • the separation on p. 35 shows in lane 7 a gel electrophoresis separation of the largest cleavage product from a sample of HS 4133/1 after complete reaction with clostridiopeptidase A.
  • the single-chain protein has a length of 486 amino acids (MG.
  • the reaction mixture contained 53 ⁇ g S 506/1 in 100 ⁇ l and had the same enzyme and buffer concentrations as the mixture for HS 4133/1. He was 80 min. incubated at 37 ° C. Every 10 to 15 minutes 10 ul samples were taken and, as in 3. 2nd described, stopped and analyzed after gel electrophoresis with a gel scanner. The results are on p. 36 shown. The product formation rate was 60 p mol / min / enzyme unit.
  • the separation on p. 35 shows in lane 5 the largest reaction product from SA 506/1 after quantitative conversion with clostridiopeptidase A.
  • the single-chain protein is 477 amino acids in length and contains the sequence that can be cleaved by collagenase
  • reaction mixture which contained 5 ⁇ g SA 302/1 and 1.0 unit of clostridiopeptidase A in 15 ⁇ l reaction buffer (as in 3.2.) was incubated at 37 ° C. for 15 h and then, as in 3.2. described, stopped by adding sample buffer and heating and analyzed by gel electrophoresis and evaluation with a gel scanner. About 0.02 p mol / min / enzyme unit of the larger reaction product had been formed. The corresponding gel electrophoresis separation is shown on page 35 in lane 9.
  • Tab. 1 Amino acid sequences of the model and fusion proteins derived from alkaline phosphatase (AP)
  • the phosphatase sequences on both sides of the collagenase interfaces are characterized by the abbreviation 'Phos' and the position numbers in parentheses of the first and last amino acid of the respective partial sequence, the numbering being based on the native pre-phosphatase (471 amino acids) from E. coli K12 relates.
  • the sequence for ACTH (1-39) is shown in the German patent application (file number P37 31 875.6).
  • Tab. 2 Product formation rates for the implementation of the modified pre-phosphatases (Tab. 1) and the pre-phosphatase / ATCH fusion proteins (see file number P 3731 875.6) with clostridiopeptidase A
  • Fig. 5 Gel electrophoresis separation of the phosphatase proteins from Table 1 and the reaction products obtained therefrom by reaction with Clostridiopeptidase A.
  • the fusion protein used in rows 2 and 3 had not been purified by gradient chromatography.
  • the lowest molecular weight band in row 3 is Gly-Pro-ACTH (1-39).
  • row 9 In addition to the clostridiopeptidase A cleavage products (ME 31 200 and 18 700), row 9 also contains products of unspecific cleavage.
  • Fig.6 Time dependence of the implementation of the proteins SA302 / 1, HS4133 / 1 and SA506 / 1 with clostridiopeptidase A

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Description

Verfahren zur Herstellung von Peptiden durch spezifische Spaltung von gentechnisch gewonnenen Fusionsproteinen mit Collagenasen Process for the production of peptides by specific cleavage of genetically engineered fusion proteins with collagenases
Die Gentechnologie hat neue Wege zur Herstellung von Peptiden und Proteinen eröffnet, welche der herkömmlichen chemischen Synthese, insbesondere bei längeren Aminosäuresequenzen, weit überlegen sind. Dadurch stehen erstmals sonst schwer zugängliche Substanzen wie Wachstumshormon, Interferone und zahlreiche Peptidhormone in ausreichenden Mengen zur Verfügung (F.A.O. Marston, Biochem. J. 240, 1-12 [1986]).Genetic engineering has opened new avenues for the production of peptides and proteins, which are far superior to conventional chemical synthesis, especially with longer amino acid sequences. This means that for the first time, otherwise difficult to access substances such as growth hormone, interferons and numerous peptide hormones are available in sufficient quantities (F.A.O. Marston, Biochem. J. 240, 1-12 [1986]).
Die gentechnischen Verfahren zur Herstellung von Polypeptiden beruhen auf der Übertragung einer geeigneten Desoxyribonukleinsäure (DNA), welche Codons für die herzustellende Aminosäuresequenz enthält, auf geeignete Wirtszellen in einer Form, welche die Replikation und die Expression der neu eingebrachten genetischen Information ermöglicht. Als Wirtszellen werden bei kleinen bis mittelgroßen Peptiden (weniger als ca. 100 Aminosäuren) vorzugsweise Bakterien wie Escherichia coli eingesetzt.The genetic engineering processes for the production of polypeptides are based on the transfer of a suitable deoxyribonucleic acid (DNA), which contains codons for the amino acid sequence to be produced, to suitable host cells in a form which enables the replication and expression of the newly introduced genetic information. Bacteria such as Escherichia coli are preferably used as host cells for small to medium-sized peptides (less than approx. 100 amino acids).
Es hat sich nun gezeigt, daß Polypeptide des genannten Größenbereiches sehr leicht von intrazellulären bakteriellen Proteasen angegriffen werden, da sie nicht durch eine ausgeprägte Tertiärstruktur geschützt sind. Dies kann zu einer starken Ausbeuteverminderung oder auch zum völligen Verlust des primären Biosyntheseproduktes führen, wenn die Expression so gelenkt wird, daß das herzustellende Polypeptid direkt in der endgültigen Form oder allenfalls mit einem zusätzlichen N-terminalen Methionin-Rest gebildet wird (sogenannte direkte Expression). Dagegen lassen sich die gewünschten Aminosäuresequenzen in der Regel in guter Ausbeute erhalten, wenn sie zunächst als sehr viel größere Vorläufermoleküle, sogenannte Fusionsproteine, synthetisiert werden.It has now been shown that polypeptides of the size range mentioned are very easily attacked by intracellular bacterial proteases, since they are not protected by a pronounced tertiary structure. This can lead to a sharp reduction in yield or even to complete loss of the primary biosynthesis product if the expression is directed in such a way that the polypeptide to be produced is formed directly in the final form or, if need be, with an additional N-terminal methionine residue (so-called direct expression) . In contrast, the desired amino acid sequences can generally be obtained in good yield if they are first synthesized as much larger precursor molecules, so-called fusion proteins.
Zur Herstellung kleinerer und mittelgroßer Peptide wird daher eine Verfahrensvariante bevorzugt, bei v/elcher durch In VitroNeukombination aus einem gut exprimierbaren und möglichst auch regulierbaren bakteriellen Gen und der DNA-Sequenz für das herzustellende Peptid ein Hybrid-Gen auf einem Plasmid-Vektor gebildet wird. Das Hybrid-Gen enthält die ursprünglichen bakteriellen Regulationselemente (Promotor, ribosomale Bindungsstelle und Terminator) sowie einen möglichst großen Teil der Codons für die N-terminalen Aminosäuren des bakteriellen Genproduktes, gefolgt von den Codons für das neue Peptid sowie einem Stopcodon, Nach Überführung des Plasmid-Vektors in eine geeignete bakterielle Wirtszelle durch Transformation steuert das Hybrid-Gen die Biosynthese eines Fusionsproteins, dessen N-terminaler Teil sich von dem entsprechenden bakteriellen Protein ableitet, während der C-terminale Teil aus der Aminosäuresequenz des herzustellenden Peptids besteht. Dieses Fusionsprotein kann leicht durch Fermentation der transformierten Zellen in großen Mengen hergestellt und isoliert werden. Verschiedene bakterielle Gene sind für die Konstruktion von Hybrid-Genen eingesetzt worden, vorzugsweise jedoch solche für die Enzyme ß-Galactosidase, ß-Lactamase, Anthranilat-Synthetase, alkalische Phosphatase und Chloramphenicol-Acetyltransferase.For the production of smaller and medium-sized peptides, a process variant is therefore preferred, in which a hybrid gene is formed on a plasmid vector by an in vitro combination of a bacterial gene which can be easily expressed and, if possible, also regulated, and the DNA sequence for the peptide to be produced. The hybrid gene contains the original bacterial regulatory elements (promoter, ribosomal binding site and terminator) and as large a part of the codons as possible for the N-terminal amino acids of the bacterial gene product, followed by the codons for the new peptide and a stop codon, after transfer of the plasmid vector into a suitable bacterial host cell by transformation, the hybrid gene controls the biosynthesis of a fusion protein, the N-terminal part of which is derived from the corresponding bacterial protein, during the C-terminal part consists of the amino acid sequence of the peptide to be produced. This fusion protein can be easily produced and isolated by fermentation of the transformed cells in large quantities. Various bacterial genes have been used for the construction of hybrid genes, but preferably those for the enzymes β-galactosidase, β-lactamase, anthranilate synthetase, alkaline phosphatase and chloramphenicol acetyltransferase.
Die letzte Stufe bei der Herstellung eines Peptids nach dem oben skizzierten Verfahren ist die exakte Spaltung des Fusionsproteins. Um diese zu ermöglichen, muß das Hybrid-Gen so konstruiert werden, daß in dem späteren Fusionsprotein zwischen dem bakteriellen Teil und der neu herzustellenden Peptidsequenz eine leicht und selektiv spaltbare Peptidbindung vorhanden ist. Die Entwicklung eines spezifischen und möglichst breit anwendbaren Verfahrens für die Spaltung von Fusionsproteinen hat sich als das größte und bisher nicht völlig zufriedenstellend gelöste Problem bei der gentechnischen Herstellung kleinerer und mittelgroßer Peptide erwiesen.The last step in the preparation of a peptide according to the procedure outlined above is the exact cleavage of the fusion protein. In order to make this possible, the hybrid gene must be constructed in such a way that an easily and selectively cleavable peptide bond is present in the later fusion protein between the bacterial part and the new peptide sequence to be produced. The development of a specific and broadly applicable process for the cleavage of fusion proteins has proven to be the biggest and so far not completely satisfactorily solved problem in the genetic engineering production of small and medium-sized peptides.
Gegenstand der vorliegenden Erfindung ist ein spezifisches und daher allgemein anwendbares Verfahren zur Spaltung von Fu sionsproteinen, welches die exakte Aminosäuresequenz des gewünschten Peptids liefert, ohne am N-terminalen Ende unerwünschte zusätzliche Aminosäuren zu hinterlassen.The present invention relates to a specific and therefore generally applicable method for splitting fu tion proteins, which provides the exact amino acid sequence of the desired peptide without leaving unwanted additional amino acids at the N-terminal end.
Die ersten gentechnisch hergestellten Peptide, wie z.B.Somatostatin (K. Itakura et al., Science 198, 1056-1063 [1977]) oder die A- und B-Kettenvon humanem Insulin (D.V. Goeddel et al., Proc. Natl. Acad. Sei, USA 76, 106-110 [1979]), waren durch Spaltung mit Bromcyan aus den entsprechenden Fusionsproteinen, welche sich von ß-Galactosidase ableiteten, freigesetzt worden. Bromcyan spaltet selektiv die Bindung zwischen Methionin und der nächsten carboxylständigen Aminosäure (Met-X, wobei X irgendeine der 20 durch den genetischen Code festgelegten Aminosäuren sein kann). Dieses Verfahren ist naturgemäß nur anwendbar für Peptide, welche selbst kein Methionin enthalten; das trifft für die drei oben genannten Peptide zu.The first genetically engineered peptides such as Somatostatin (K. Itakura et al., Science 198, 1056-1063 [1977]) or the A and B chains of human insulin (DV Goeddel et al., Proc. Natl. Acad. Sei, USA 76, 106-110 [1979]), had been released by cleavage with cyanogen bromide from the corresponding fusion proteins which were derived from β-galactosidase. Bromocyan selectively cleaves the bond between methionine and the next carboxyl amino acid (Met-X, where X can be any of the 20 amino acids specified by the genetic code). Naturally, this method can only be used for peptides which themselves do not contain methionine; this applies to the three peptides mentioned above.
Gleichartige Einschränkungen gelten für alle Verfahren zur Spaltung von Fusionsproteinen, welche auf der Erkennung einer einzelnen Aminosäure beruhen. So sind Fusionsproteine hergestellt worden, welche die Aminosäuren Lysin, Arginin oder Glutaminsäure als Verknüpfung zwischen dem N-terminalen und dem C-terminalen Teil enthalten. Zur Spaltung wurden die Enzyme Trypsin (J. Shine et al., Nature 285, 456-461 [1980]) und Clostripain (A.D. Bennett et al., EPA 0131363 ), welche beide die Bindung zwischen Lys-X und Arg-X öffnen sowie Endoproteinase Glu-C (A.D. Bennett et al., EPA 0131363), welche die Bindung Glu-X spaltet, verwendet. Endoproteinase Lys-C (G. Allen et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, S. 162, Abstr. 19.01.03), die spezifisch nach Lysin spaltet, ist ebenfalls eingesetzt worden.Similar restrictions apply to all methods for cleaving fusion proteins which are based on the recognition of a single amino acid. Thus fusion proteins have been produced which contain the amino acids lysine, arginine or glutamic acid as a link between the N-terminal and the C-terminal part. For the cleavage, the enzymes were trypsin (J. Shine et al., Nature 285, 456-461 [1980]) and clostripain (AD Bennett et al., EPA 0131363), both of which open the bond between Lys-X and Arg-X and endoproteinase Glu-C (AD Bennett et al., EPA 0131363), which cleaves the Glu-X bond. Endoproteinase Lys-C (G. Allen et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, p. 162, Abstr. 19.01.03), which specifically cleaves for lysine, has also been used.
Sehr viel spezifischer und damit breiter anwendbar sind Verfahren, die eine spezifische Bindung innerhalb einer genau definierten Sequenz aus mehreren Aminosäuren spalten. So konnten P.R. Szoka et al. (DNA 5, 11-20 [1986]) ein Fusionsprotein, in dem eine Teilsequenz aus Anthranilat-Synthetase sowie Rinder-Wachstumshormon über die Sequenz Asp-Pro miteinander verknüpft sind, durch Einwirkung von Säure in die beiden Bestandteile zerlegen, weil die Bindung Asp-Pro sehr säurelabil ist. Neben der Tatsache, daß man bei der Einwirkung von Säure auch mit der Spaltung weiterer Peptidbindungen als Nebenreaktion rechnen muß, hat dieses Verfahren vor allen den Nachteil, daß das Prolin aus der Kopplungssequenz am N-terminalen Ende des Peptids übrig bleibt, was in vielen Fällen nicht tolerabel ist. Die gleiche Einschränkung muß man bei der Umsetzung mit Renin machen, welches verwendet worden ist, um in einem Fusionsprotein innerhalb der Sequenz Pro-Phe-His-Leu-Leu-Val-Tyr selektiv die Bindung zwischen den beiden Leucin-Resten zu spalten (J.S. Boger et al., EPA 0163573.). In diesem Fall bleiben drei zusätzliche und häufig unerwünschte N-terminale Aminosäuren übrig.Processes that cleave a specific bond within a precisely defined sequence of several amino acids are much more specific and therefore more widely applicable. So P.R. Szoka et al. (DNA 5, 11-20 [1986]) a fusion protein, in which a partial sequence from anthranilate synthetase and bovine growth hormone are linked to one another via the sequence Asp-Pro, decompose into the two components by the action of acid, because the binding Asp -Pro is very acid labile. In addition to the fact that one must also expect the cleavage of further peptide bonds as a side reaction when exposed to acid, this method has the disadvantage above all that the proline remains from the coupling sequence at the N-terminal end of the peptide, which in many cases is not tolerable. The same restriction must be made when reacting with renin, which has been used to selectively cleave the bond between the two leucine residues in a fusion protein within the Pro-Phe-His-Leu-Leu-Val-Tyr sequence (JS Boger et al., EPA 0163573.). In this case, three additional and often undesirable N-terminal amino acids remain.
Faktor Xa, eine Plasmaprotease, deren normale Funktion die Umwandlung von Prothrombin in Thrombin ist, ist von K. Nagai und H.C. Thtfgersen (Nature 309, 810-812 [1984]) verwendet worden, um humanes ß-Globin aus einem Fusionsprotein freizusetzen, des sen N-terminaler Teil aus 31 Aminosäuren des λcII-Proteins bestand. In ähnlicher Weise haben T. Imai et al. (J. Biochem. 100, 425-432 [1986]) das Enzym eingesetzt, um humanes Prorenin über ein Fusionsprotein mit 9 zusätzlichen N-terminalen Aminosäuren herzustellen. In beiden Fällen war als Übergang zwischen N- und C-terminalem Bereich die Sequenz Ile-Glu-Gly-Arg-X vorhanden, die vom Faktor Xa spezifisch erkannt und an der Bindung zwischen Arg und X gespalten wird (wobei X die erste N-terminale Aminosäure des humanen Proteins ist). Faktor Xa scheint daher die Forderungen zu erfüllen, die man an ein Enzym stellen muß, welches für die Spaltung von Fusionsproteinen eingesetzt werden soll. Allerdings sind auch zwei Fälle berichtet worden, in denen eine Spaltung von Fusionsproteinen, welche die obige Erkennungssequenz enthielten, mit Faktor Xa nicht möglich war (G. Allen et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, S. 162, Abstr. 19.01.03; H. Mayer et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, S. 285, Abstr. 05.01.50). Eine generelle Anwendung dieses Verfahrens ist daher nicht mögl ich.Factor Xa, a plasma protease whose normal function is the conversion of prothrombin to thrombin, has been used by K. Nagai and HC Thtfgersen (Nature 309, 810-812 [1984]) to release human β-globin from a fusion protein Its N-terminal part consisted of 31 amino acids of the λcII protein. Similarly, T. Imai et al. (J. Biochem. 100, 425-432 [1986]) used the enzyme to produce human prorenin via a fusion protein with 9 additional N-terminal amino acids. In both cases, the sequence Ile-Glu-Gly-Arg-X was present as a transition between the N- and C-terminal region, which is specifically recognized by factor Xa and cleaved at the bond between Arg and X (where X is the first N- terminal amino acid of human protein). Factor Xa therefore seems to meet the requirements that must be placed on an enzyme which is to be used for the cleavage of fusion proteins. However, two cases have also been reported in which a cleavage of fusion proteins containing the above recognition sequence was not possible with factor Xa (G. Allen et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, P. 162, abstr. 19.01.03; H. Mayer et al., Biol. Chem. Hoppe-Seyler 367, Suppl. Aug. 1986, p. 285, abstr. 05.01.50). A general application of this method is therefore not possible.
Ähnliche Einschränkungen muß man für das Enzym Enterokinase machen, welches in der Sequenz (Asp)4-Lys-X die Bindung zwischen Lys und X spaltet. R.M. Belagaje et al. (DNA 3, 120 [1984]) haben zwar mit diesem Enzym menschliches Wachstumshormon aus einem Fusionsprotein freigesetzt, in welchem sich diese Sequenz nicht weit vom N-terminalen Ende entfernt befindet, ähnlich wie in Trypsinogen, dem natürlichen Substrat der Enterokinase.Similar restrictions have to be made for the enzyme enterokinase, which cleaves the bond between Lys and X in the sequence (Asp) 4 -Lys-X. RM Belagaje et al. (DNA 3, 120 [1984]) have released human growth hormone with this enzyme from a fusion protein in which this sequence is not far from the N-terminal end, similar to that in Trypsinogen, the natural substrate of enterokinase.
Versuche, Fusionsproteine, in denen alkalische Phosphatase und adrenocorticotropes Hormon über die genannte Sequenz verknüpft waren, mit Enterokinase zu spalten, waren jedoch nicht erfolgreich.However, attempts to cleave fusion proteins in which alkaline phosphatase and adrenocorticotropic hormone were linked via the sequence mentioned with enterokinase were unsuccessful.
In EP 20290 wird ein Verfahren beschrieben, das die hohe Spezifität von Collagenasen zur Spaltung von Fusionsproteinen entsprechender Struktur ausnutzt.EP 20290 describes a method which uses the high specificity of collagenases to cleave fusion proteins of corresponding structure.
Collagenasen erkennen die Sequenz Pro-Y-Gly-Pro, wobei Y jede beliebige der 20 Aminosäuren des genetischen Codes sein kann, und spalten zwischen Y und Gly. Aus einem Fusionsprotein, welches diese Sequenz enthält, wird ein Peptid freigesetzt, welches am N-terminalen Ende noch die aus der Collagenase-Sequenz stammenden Aminosäuren Gly und Pro enthält. Diese können als Dipeptid mit einem weiteren Enzym, der Postprolin-Dipeptidyl-Aminopeptidase (PPDA), entfernt werden.Collagenases recognize the sequence Pro-Y-Gly-Pro, where Y can be any of the 20 amino acids of the genetic code, and split between Y and Gly. A fusion protein which contains this sequence releases a peptide which at the N-terminal end also contains the amino acids Gly and Pro derived from the collagenase sequence. These can be removed as a dipeptide with another enzyme, the postproline dipeptidyl aminopeptidase (PPDA).
Versuche zur Spaltung von Peptiden und Proteinen, welche die Sequenz Pro-Y-Gly-Pro enthalten, mit Clostridiopeptidase A, einer Collagenase aus Clostridium histolyticum (EC 3.4.24.3), haben nun gezeigt, daß die Anwendung der oben beschriebenen Reaktionsfolge zur Herstellung von Peptiden über Fusionsproteine nur in sehr begrenztem Maße möglich ist. Zwar konnten M. Töpert et al. (Mitteilung beim 14. FEBS-Meeting [1981]) zeigen, daß sich ein relativ kleines, halbsynthetisches Modellpeptid, und zwar die VerbindungAttempts to cleave peptides and proteins which contain the sequence Pro-Y-Gly-Pro with Clostridiopeptidase A, a collagenase from Clostridium histolyticum (EC 3.4.24.3), have now shown that the use of the reaction sequence described above for the production of Peptides via fusion proteins are only possible to a very limited extent. M. Töpert et al. (Communication at the 14th FEBS meeting [1981]) show that there is a relatively small, semi-synthetic model peptide, namely the compound
Cbo-Gly-Pro-Leu-Gly-Pro-Insulin-A-Kette (Rind) entsprechend dem oben dargestellten Schema mit Clostridiopeptidase A und PPDA umsetzen läßt, wobei die erwarteten Reaktionsprodukte in guter Ausbeute erhalten wurden. Ähnliche Ergebnisse erhielten E. Wünsch et al. (Hoppe-Seyler ' s Z. Physiol. Chem. 362, 1285-1287 [l981]) bei der Umsetzung eines anderen Modellpeptids. Dagegen ließen sich von vier natürlichen Proteinen mit der Sequenz Pro-Y-Gly-Pro nur zwei (Neurophysin II und Rinderkatalase) mit Clostridiopeptidase A spezifisch spalten, und dies auch nur nach Umwandlung der Cystein-Seitenketten in die S-Sulfonate. Trotz eines relativ großen Enzymüberschusses konnte jedoch keine quantitative Umsetzung erreicht werden (M. Töpert et al., Mitteilung beim 14. FEBS-Meeting [l981]).Cbo-Gly-Pro-Leu-Gly-Pro Insulin A Chain (Bovine) can be reacted with clostridiopeptidase A and PPDA according to the scheme shown above, the expected reaction products being obtained in good yield. E. Wünsch et al. (Hoppe-Seyler's Z. Physiol. Chem. 362, 1285-1287 [1981]) in the implementation of another model peptide. In contrast, only four of four natural proteins with the sequence Pro-Y-Gly-Pro (neurophysin II and bovine catalase) could be cleaved specifically with clostridiopeptidase A, and only after the cysteine side chains had been converted into the S-sulfonates. Despite a relatively large excess of enzymes, however, no quantitative conversion could be achieved (M. Töpert et al., Communication at the 14th FEBS meeting [1981]).
In der deutschen Patentanmeldung (Aktenzeichen P 3731 875.6) wird ein Verfahren zur Herstellung von adrenocorticotropem Hormon über Fusionsproteine beschrieben, die sich von der alkalischen Phosphatase aus E.coli ableiten. Auch in diesem Fall wurde gefunden, daß Fusionsproteine mit einer Tetrapeptidsequenz Pro-Y-Gly-Pro zwischen bakterieller Proteinsequenz und Zielpeptid, wobei Y jede der 20 Aminosäuren des genetischen Codes bedeutet, sich zwar spezifisch an der vorbestimmten Stelle mit Clostridiopeptidase A spalten lassen, jedoch war die Reaktionsgeschwindigkeit sehr niedrig.The German patent application (file number P 3731 875.6) describes a process for the production of adrenocorticotropic hormone via fusion proteins which are derived from the alkaline phosphatase from E. coli. In this case, too, it was found that fusion proteins with a tetrapeptide sequence Pro-Y-Gly-Pro between bacterial protein sequence and target peptide, where Y is each of the 20 amino acids of the genetic code, can be cleaved specifically at the predetermined position with clostridiopeptidase A, however the response speed was very slow.
J. Germino und D. Bastia (Proc. Natl. Acad. Sei. USA 81, 4692-4696 [l984]) haben ein Fusionsprotein hergestellt, welches als Bindeglied zwischen dem N- und C-terminalen Bereich eine Sequenz aus ca. 60 Aminosäuren aus Proα-2-Collagen enthielt. Die- ses Protein ließ sich mit Collagenasen ähnlich gut spalten wie das entsprechende Collagen selbst. Es ist jedoch nicht bekannt, an welchen Stellen innerhalb der Sequenz von ungefähr 60 Aminosäuren das Fusionsprotein genau gespalten wird. Es zeigte sich außerdem, daß ein mehr oder weniger großer Rest dieser Sequenz noch am N-terminalen Ende des C-terminalen Spaltproduktes vorhanden ist. Daher ist diese Verfahrensvariante zur Herstellung von Polypeptiden mit genau vorgegebenem N-terminalen Ende ungeeignet.J. Germino and D. Bastia (Proc. Natl. Acad. Sci. USA 81, 4692-4696 [l984]) have produced a fusion protein which, as the link between the N- and C-terminal region, has a sequence of approx. 60 amino acids from Proα-2 collagen contained. The- This protein could be cleaved with collagenases as well as the corresponding collagen itself. However, it is not known at which locations within the sequence of approximately 60 amino acids the fusion protein is cleaved exactly. It was also found that a more or less large residue of this sequence is still present at the N-terminal end of the C-terminal cleavage product. This process variant is therefore unsuitable for the production of polypeptides with a precisely specified N-terminal end.
Die vorliegende Erfindung besteht in einem Verfahren zur Herstellung von Zielpeptiden, das dadurch gekennzeichnet ist, daß man aus einem Fusionsprotein der Formel I,The present invention consists in a process for the preparation of target peptides, which is characterized in that a fusion protein of the formula I,
H2N-Z1-X-(Pro-Y-Gly)n-Pro-Z2-COOH (I), in der n ≥ 2 ist,H 2 NZ 1 -X- (Pro-Y-Gly) n -Pro-Z 2 -COOH (I), in which n ≥ 2,
X und Y jede der 20 durch den genetischen Code festgelegten Aminosäuren darstellt, Z 1 eine bakterielle Aminosäuresequenz undX and Y represent each of the 20 amino acids defined by the genetic code, Z 1 represents a bacterial amino acid sequence and
Z2 das Zielpeptid aus beliebigen Aminosäuren des genetischen Codes bedeuten,Z 2 denotes the target peptide from any amino acids of the genetic code,
die C-terminal auf die Aminosäuresequenz -X-(Pro-Y-Gly)n-Pro-, in der X und Y jede genetisch kodierbare Aminosäure und n ≥ 2 bedeuten, folgende Proteinsequenz enzymatisch mit Collagenase und Postprolindipeptidylaminopeptidase (PPDA) abspaltet. Die Erfindung betrifft außerdem Fusionsproteine der Formel I und Verfahren zu ihrer Herstellung gemäß Ansprüchen 1 - 6, sowie Genstrukturen, die für Fusionsproteine der Formel I kodieren und Plasmide oder vergleichbare Vektoren (z.B. Phagenvektoren), die Codons für repetitive CollagenaseSchnittstellen enthalten und damit zur Herstellung von Fusionsproteinen der Formel I geeignet sind.the C-terminal to the amino acid sequence -X- (Pro-Y-Gly) n -Pro-, in which X and Y are each genetically encodable amino acid and n ≥ 2, enzymatically cleaves the following protein sequence with collagenase and postproline dipeptidylaminopeptidase (PPDA). The invention also relates to fusion proteins of the formula I and processes for their preparation according to claims 1-6, and to gene structures which code for fusion proteins of the formula I and plasmids or comparable vectors (for example phage vectors) which contain codons for repetitive collagenase interfaces and thus for the production of Fusion proteins of formula I are suitable.
Als bakterielle Aminosäuresequenz Z1 kommen alkalische Phosphatase, Anthranilat-Synthetase, β-Lactamase, β-Galactosidase, Chloramphenicol-Acetyltransferase etc. in Frage.As bacterial amino acid sequence Z 1 , alkaline phosphatase, anthranilate synthetase, β-lactamase, β-galactosidase, chloramphenicol acetyl transferase etc. come into question.
Das nachstehende Reaktionsschema erläutert die proteolytische Spaltung von Fusionsproteinen mit Collagenase und Postprolin-Dipeptidyl-Aminopeptidase (PPDA): The following reaction scheme explains the proteolytic cleavage of fusion proteins with collagenase and postproline dipeptidyl aminopeptidase (PPDA):
Für X und Y kann jede der im genetischen Code festgelegten 20 Aminosäuren stehen, n bedeutet bevorzugt 2 bis 10.X and Y can be any of the 20 amino acids specified in the genetic code, n preferably means 2 to 10.
Der Vorteil des neuen Verfahrens zur Spaltung von Fusionsproteinen besteht gegenüber dem Verfahren aus EP 20290 darin, daß die Umsetzungsgeschwindigkeit z.B. mit Clostridiopeptidase um Größenordnungen gesteigert werden kann. Fusionsproteine, die die Erkennungssequenz für Collagenase in repetitiver Form enthalten, lassen sich unvergleichlich viel besser spalten als die entsprechenden Fusionsproteine mit einer einfachen Spaltstelle für Collagenasen. Zur Veranschaulichung des erfindungsgemäßen Verfahrens werden in den Beispielen 1-3 die Herstellung und enzymatische Spaltung von 3 Modellproteinen beschrieben, welche verschiedene, durch Collagenasen spaltbare Sequenzen enthalten. Die Aminosäuresequenzen dieser Proteine sind in Tab. 1 zusammengefaßt. Sie leiten sich von der Präphosphatase aus E.coli K 12 ab, einem Zwischenprodukt der Biosynthese von alkalischer Phosphatase, welche aus 471 Aminosäuren besteht. Durch Abspalten einer N-terminalen Signalsequenz von 22 Aminosäuren wird die Präphosphatase in das aktive Enzym überführt. Dagegen wird bei den modifizierten Phosphatasen die Signalsequenz von 22 Aminosäuren nicht in nennenswertem Umfang abgespalten.The advantage of the new process for cleaving fusion proteins over the process from EP 20290 is that the rate of conversion can be increased by orders of magnitude, for example with clostridiopeptidase. Fusion proteins that contain the recognition sequence for collagenase in repetitive form are incomparably easier to cleave than the corresponding fusion proteins with a simple cleavage site for collagenases. To illustrate the process according to the invention, examples 1-3 describe the preparation and enzymatic cleavage of 3 model proteins which contain different sequences which can be cleaved by collagenases. The amino acid sequences of these proteins are summarized in Table 1. They are derived from the prephosphatase from E.coli K 12, an intermediate product in the biosynthesis of alkaline phosphatase, which consists of 471 amino acids. The pre-phosphatase is converted into the active enzyme by cleaving off an N-terminal signal sequence of 22 amino acids. In contrast, the signal sequence of 22 amino acids is not split off to any appreciable extent in the modified phosphatases.
Die Aminosäuresequenz der alkalischen Phosphatase (R.A. Bradshaw et al., Proc. Natl. Acad. Sei. USA 81, 4692-4696 [1981]) sowie die Nukleotidsequenz des entsprechenden Gens aus E.coli K12 (phoA) sind bekannt (M. Simonis, Dissertation, Freie Universität Berlin [1983]; C.N. Chang et al., Gene 44, 121-125 [1986]); Vektoren, die für die Konstruktion von Fusionsproteinen auf der Basis von alkalischer Phosphatase verwendet werden können, sind ebenfa beschrieben worden (W. Boidol et al., Mol. Gen. Genet. 185, 510512 [1982]; W. Boidol, Dissertation, Technische Universität Berlin [1984]). Ausgehend von einem dieser Plasmide, welches die Bezeichnung pSB94 trägt, wurden mit an sich bekannten Methoden der in vitro-Neukombination und DNA-Klonierung (T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA [1982]) modifizierte Vektoren hergestellt, welche nach Überführung in geeignete E.coli-Wirtszellen die Bio synthese der in Tab. 1 dargestellten Proteine bewirken. Dazu wurden in zwei Positionen des phoA-Gens auf pSB94, nämlich in die NcoI- bzw. in die Sphl-Sequenz, Oligonukleotide eingefügt, welche in den modifizierten Genen Codons für die durch Collagenasen erkennbaren Aminosäuresequenzen bilden. Restriktionskarten von pSB94 und der davon abgeleiteten Plasmide pSA302, pSA506 und pHS4133 sind in den Abb. 1-4 dargestellt.The amino acid sequence of alkaline phosphatase (RA Bradshaw et al., Proc. Natl. Acad. Sci. USA 81, 4692-4696 [1981]) and the nucleotide sequence of the corresponding gene from E. coli K12 (phoA) are known (M. Simonis , Dissertation, Freie Universität Berlin [1983]; CN Chang et al., Gene 44, 121-125 [1986]); Vectors which can be used for the construction of fusion proteins based on alkaline phosphatase have also been described (W. Boidol et al., Mol. Gen. Genet. 185, 510512 [1982]; W. Boidol, Dissertation, Technische University of Berlin [1984]). Starting from one of these plasmids, which bears the designation pSB94, known methods of in vitro recombination and DNA cloning (T. Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) were used [1982]) produced modified vectors which, after transfer into suitable E. coli host cells, the Bio effect synthesis of the proteins shown in Tab. 1. For this purpose, oligonucleotides were inserted in two positions of the phoA gene on pSB94, namely in the NcoI or in the Sphl sequence, which form codons in the modified genes for the amino acid sequences recognizable by collagenases. Restriction maps of pSB94 and the plasmids pSA302, pSA506 and pHS4133 derived therefrom are shown in FIGS. 1-4.
Die Ergebnisse der Umsetzung der 3 Modellproteine mit Clostridiopeptidase A sind auf Seite 35 und 36 sowie in Tab. 2 dargestellt. S. 35 zeigt eine gelelektrophoretisehe Trennung, die die Entstehu von Spaltprodukten des erwarteten Molekulargewichtes und damit die spezifische Spaltung an der vorbestimmten Stelle belegt. Auf Seite 36 ist die Abhängigkeit der Produktbildung von der Inkubationszeit und der eingesetzten Enzymmenge dargestellt. Die aus den Kurven ermittelten Produktbildungsraten, die in Tab. 2 zusammengefaßt sind, machen deutlich, in welchem Umfang eine Doppelschnittstelle (HS 4133/1) und erst recht eine fünff-ache-Schnittstelle (SA 506/1) die Reaktionsfähigkeit gegenüber Collagenasen im Vergleich zu einer einfachen Schnittstelle (SA 302/1) erhöhen.The results of the implementation of the 3 model proteins with clostridiopeptidase A are shown on pages 35 and 36 and in Table 2. P. 35 shows a gel electrophoresis separation, which shows the formation of cleavage products of the expected molecular weight and thus the specific cleavage at the predetermined position. The dependence of product formation on the incubation time and the amount of enzyme used is shown on page 36. The product formation rates determined from the curves, which are summarized in Tab. 2, make clear to what extent a double interface (HS 4133/1) and especially a five-axis interface (SA 506/1) compare the reactivity to collagenases to a simple interface (SA 302/1).
In όir deutschen Patentanmeldung (Aktenzeichen P 37 31 875.6 ). deren Gegenstand ein Verfahren zur Herstellung von adrenocorticotropem Hormon und davon abgleiteten Peptiden ist, werden Fusionsproteine beschrieben, in denen die Hormonsequenz über eine einfache bzw. repetitive Collagenase-Schnil-tstellen an Teilsequenzen der alkalischen Phosphatase aus E.coli gekoppelt sind. Die dort beschrie benen Versuche zur Umsetzung dieser Fusionsproteine mit Clostridiopeptidase A bestätigen in vollem Umfang die in der vorliegenden Erfindung beschriebenen Resultate. Die Produktbildungsraten der wichtigsten ACTH-Fusionsproteine sind ebenfalls in Tab. 2 enthalten. Während aus dem Protein mit einer einfachen Collagenase-Schnittstelle (SA186/1) das Peptidhormon nur unvollständig und unter Bedingungen freigesetzt werden konnte, die für eine praktische präparative Anwendung ungeeignet sind, wurde das entsprechende Protein mit einer doppelten Schnittstelle (SA343/1) bereits um einen Faktor von mindestens 103 besser gespalten. Durch Verwendung einer fünffachen bzw. einer achtfachen Schnittstelle (SA341/1 bzw. SA360/1) wurde die Reaktionsgeschwindigkeit weiter erhöht. Die Sequenz des ACTH-Fusionsproteins SA 341/1 ist in Tab. 1 dargestellt. Die Trennung auf Seite 35 zeigt in Spur 3 die Produkte der Umsetzung von SA341/1 mit Clostridiopeptidase A.In όir German patent application (file number P 37 31 875.6). The subject of which is a process for the production of adrenocorticotropic hormone and peptides derived therefrom, fusion proteins are described in which the hormone sequence is coupled to partial sequences of the alkaline phosphatase from E. coli via a simple or repetitive collagenase interface. Described there Attempts to implement these fusion proteins with clostridiopeptidase A fully confirm the results described in the present invention. The product formation rates of the most important ACTH fusion proteins are also shown in Tab. 2. While the protein with a simple collagenase interface (SA186 / 1) could only be released incompletely and under conditions that are unsuitable for practical preparative use, the corresponding protein with a double interface (SA343 / 1) was already released split a factor of at least 10 3 better. The reaction rate was further increased by using a five-fold or eight-fold interface (SA341 / 1 or SA360 / 1). The sequence of the ACTH fusion protein SA 341/1 is shown in Table 1. The separation on page 35 shows in lane 3 the products of the reaction of SA341 / 1 with clostridiopeptidase A.
Die Umsetzung mit PPDA entsprechend dem bereits oben angegebenen Schema kann bei der Verwendung von repetitiven Collagenase-Schnittstellen nur dann zu dem autentischen Peptid mit der exakten Aminosäuresequenz am N-terminalen Ende führen, wenn innerhalb der repetitiven Schnittstelle auch die Y-Gly-Bindung gespalten wird, die dem N-terminalen Ende des Peptids am nächsten liegt. Analytische Untersuchungen durch Gelelektrophorese und HPLC ließen keine Heterogenität der Spaltprodukte aus Fusionsproteinen mit repetitiven Collagenase- Schnittstellen erkennen. Dies weist darauf hin, daß bei der Umsetzung mit Clostridiopeptidase A alle Y-Gly- Bindungen der repetitiven Sequenz geschnitten werden. Wie in der deutschen Patentanmeldung (Aktenzeichen P3731 875.6 ) beschrieben, wurde dies am Beispiel des entsprechenden Spaltproduktes aus SA 341/1 durch Bestimmung einer N-terminalen Partialsequenz bestätigt: der Edman-Abbau ergab die für die ersten sieben Aminosäuren von Gly-Pro-ACTH erwartete Sequenz. Offenbar werden durch Spalten einer ersten Bindung innerhalb einer repetitiven Sequenz die weiteren Spaltstellen durch Freilegung und bessere Zugänglichkeit so aktiviert, daß sie sehr viel rascher reagieren als die erste Spaltstelle, so daß Zwischenprodukte unvollständiger Spaltung nicht zu erfassen sind.When using repetitive collagenase cleavages, the reaction with PPDA according to the scheme already given can only lead to the authentic peptide with the exact amino acid sequence at the N-terminal end if the Y-Gly bond is also cleaved within the repetitive cleavage site closest to the N-terminal end of the peptide. Analytical investigations by gel electrophoresis and HPLC showed no heterogeneity of the cleavage products from fusion proteins with repetitive collagenase interfaces. This indicates that all Y-Gly bonds of the repetitive sequence are cut in the reaction with clostridiopeptidase A. As described in the German patent application (file number P3731 875.6), this was done using the example of the corresponding cleavage product from SA 341/1 was confirmed by determining an N-terminal partial sequence: Edman degradation gave the sequence expected for Gly-Pro-ACTH for the first seven amino acids. Apparently, by splitting a first bond within a repetitive sequence, the other cleavage sites are activated by exposure and better accessibility so that they react much more quickly than the first cleavage site, so that intermediate products of incomplete cleavage cannot be detected.
Durch die Verwendung repetitiver Collagenase-Schnittstellen wird die Spezifität des Verfahrens deutlich erhöht, und selbst Peptide, die zufällig die Sequenz Pro-X-Gly-Pro enthalten, könnten wegen der geringen Reaktionsfähigkeit dieser Sequenz unter den hier angewendeten Bedingungen hergestellt werden. Damit ist das Verfahren zur Herstellung praktisch jeden beliebigen Peptids geeignet, und auch in der Wahl der Wirts-/Vektor-Systeme sowie für die Verwendung anderer Collagenasen und Aminopeptidyl-Dipeptidasen ähnlicher Spezifität gibt es keinerlei Einschränkungen.The use of repetitive collagenase interfaces significantly increases the specificity of the method, and even peptides that happen to contain the sequence Pro-X-Gly-Pro could be produced under the conditions used here because of the low reactivity of this sequence. The method is therefore suitable for the production of virtually any peptide, and there are no restrictions in the choice of host / vector systems or in the use of other collagenases and aminopeptidyl dipeptidases of similar specificity.
Das erfindungsgemäße Verfahren läßt sich natürlich auch in einem Schritt, d.h. bei gleichzeitiger Umsetzung mit Collagenase und PPDA, durchführen.The method according to the invention can of course also be carried out in one step, i.e. with simultaneous implementation with collagenase and PPDA.
Am 16.07.1979 wurde bei der American Type Culture Collection (ATCC) in Rockeville, Md., USA hinterlegt (Hinterlegungsnummer) :On July 16, 1979, the American Type Culture Collection (ATCC) in Rockeville, Md., USA deposited (deposit number):
Plasmid pBR 322 (ATCC 40015) . Am 16.07.1979 wurde bei der DSM, Göttingen hinterlegt:Plasmid pBR 322 (ATCC 40015). On July 16, 1979 the following was deposited with the DSM, Göttingen:
Escherichia coli SB 44 (DSM 1606). Seit dem 26.11.1973 liegt bei der DSM eine Hinterlegung vor für:Escherichia coli SB 44 (DSM 1606). Since November 26, 1973, the DSM has deposited for:
Escherichia coli K 12 Wildtyp (DSM 498). B E I S P I E L EEscherichia coli K 12 wild type (DSM 498). EXAMPLES
1. Herstellung von neukombinierten Plasmiden mit Genen für die Phosphataseproteine SA 302/1, SA 506/1 und HS 4133/11. Preparation of recombined plasmids with genes for the phosphatase proteins SA 302/1, SA 506/1 and HS 4133/1
Die Biosynthese der drei Proteine wird in entsprechend transformierten Bakterienzellen durch die neukombinierten Plasmide pSA302, pSA506 und pHS4133 determiniert. Diese Plasmide sind aus dem Vektorplasmid pSB94 entstanden. pSB94 wurde aus dem Plasmid pSB53 (hinterlegt am 16.7.79 unter der Nr. ATCC 40020) nach einem beschriebenen Verfahren (W. Boidol, Dissertation, Technische Universität Berlin [1984], siehe auch US-Patent Nr. 4,375,514) abgeleitet.The biosynthesis of the three proteins is determined in appropriately transformed bacterial cells by the newly combined plasmids pSA302, pSA506 and pHS4133. These plasmids originated from the vector plasmid pSB94. pSB94 was derived from the plasmid pSB53 (deposited on July 16, 1979 under the number ATCC 40020) according to a described method (W. Boidol, dissertation, Technical University Berlin [1984], see also US Pat. No. 4,375,514).
Herstellung des Plasmids DSB94 [W. Boidol, Dissertation TU-Berlin D83/FB13, Nr. 158 (1984)]Preparation of the plasmid DSB94 [W. Boidol, dissertation TU-Berlin D83 / FB13, No. 158 (1984)]
Ausgehend vom Plasmid pSB53 [EP-Patent 23882. ATCC 40020 (16.7.79)] wurde das Plasmid pSB94 wie folgt hergestellt:Starting from the plasmid pSB53 [EP patent 23882. ATCC 40020 (16.7.79)], the plasmid pSB94 was prepared as follows:
In einer Reaktionsmischung bestehend aus 60μl pSB53 (205μg/ml), 60μl Reaktionspuffer (20mM Tris HCl, 20mM MgCl 150mM NaCl, 20mM Mercaptoäthanol, pH 7.5), 18μl Rinderserumalbumin (1mg/ml), 5μl Sal I, 5μl Xho I und 12μl H2O wurde die pSB53-DNS 3 Stunden bei 37°C inkubiert.In a reaction mixture consisting of 60μl pSB53 (205μg / ml), 60μl reaction buffer (20mM Tris HCl, 20mM MgCl 150mM NaCl, 20mM mercaptoethanol, pH 7.5), 18μl bovine serum albumin (1mg / ml), 5μl Sal I, 5μl Xho I and 12μl H 2 O, the pSB53 DNA was incubated at 37 ° C for 3 hours.
Die Restriktionsenclσnukleasen Sal I und Xho I wurden von der Fa. Biolabs (USA) bezogen.The restriction enucleases Sal I and Xho I were obtained from Biolabs (USA).
Die Reaktionsbedingungen, die zu einer quantitativen Spaltung von pSB53 (MG-Bestimmung mittels Gel-Elektrophorese) in Fragmente von 2255 bp und 6454 bp (MG-Bestimmung mittels Sequenzanalyse) führen, wurden in Vorversuchen ermittelt. Die Trennung der beiden Fragmente erfolgte durch AgaroseGelelektrophorese.The reaction conditions leading to a quantitative cleavage of pSB53 (MG determination by means of gel electrophoresis) into fragments of 2255 bp and 6454 bp (MG determination by means of sequence analysis) were determined in preliminary experiments. The two fragments were separated by agarose gel electrophoresis.
Das 6454 bp-Fragment wurde aus einem 0,7 % Agarosegel durch Elektroelution isoliert [T. Maniatis, E. Fritsch, J. Sambrock; Molecular Cloning, 164-165, Cold Spring Harbor Lab. New York (1982)] und in einem Ligaseansatz über die komplementären 5'- überhängenden Einzelstrangenden zu Ringmolekülen verknüpft. Die Ligasereaktion mit T4-Ligase (Biolabs, USA) erfolgte in einem Reaktionspuffer mit 50mM TrisHCl, 10mM MgCl2, 20mM Dithiothreitol, 50μg/ml Rinderserumalbumin, 1mM ATP, pH 7.8. Der Ligaseansatz (100μl Volumen) wurde 16 Stunden bei 13°C inkubiert.The 6454 bp fragment was isolated from a 0.7% agarose gel by electroelution [T. Maniatis, E. Fritsch, J. Sambrock; Molecular Cloning, 164-165, Cold Spring Harbor Lab. New York (1982)] and linked in a ligase approach via the complementary 5'-overhanging single strand ends to form ring molecules. The ligase reaction with T4 ligase (Biolabs, USA) was carried out in a reaction buffer with 50mM TrisHCl, 10mM MgCl 2 , 20mM dithiothreitol, 50μg / ml bovine serum albumin, 1mM ATP, pH 7.8. The ligase mixture (100 μl volume) was incubated at 13 ° C. for 16 hours.
Die Reaktion wurde durch Phenolextraktion gestoppt und die DNS aus der wässrigen Phase nach dreimaliger Ätherextraktion mit Äthanol gefällt. Nach Zentrifugation wurde der DNS-Niederschlag in 50μl DNS-Puffer (45mM TnsHCl, 0,1mM EDTA, pH 7.9) gelöst und zur Transformation kompetenter Zellen des Stammes Escherichia coli SB44 eingesetzt [200μl kompetente Zellen +25μl der DNS aus dem Ligaseansatz].The reaction was stopped by phenol extraction and the DNA was precipitated from the aqueous phase after three ether extractions with ethanol. After centrifugation, the DNA precipitate was dissolved in 50μl DNA buffer (45mM TnsHCl, 0.1mM EDTA, pH 7.9) and used to transform competent cells of the Escherichia coli SB44 strain [200μl competent cells + 25μl DNA from the ligase batch].
Die Herstellung von kompetenten Zellen sowie Transformationsbedingungen entsprechen den in EP 23882 beschriebenen Verfahren und Bedingungen.The production of competent cells and transformation conditions correspond to the methods and conditions described in EP 23882.
Aus den erhaltenen Transformanten (ampicillinresistente Zellen) wurde Plasmid-DNS isoliert und durch Restriktionsanalyse die Struktur des Plasmids bestätigt. In Abb. 1 ist eine Restriktionskarte von pSB94 dargestellt. (Die Abstände zwischen den einzelnen Restriktionssequenzen sind in Basenpaaren (Bp) angegeben, die Gesamtgröße beträgt 6454 Bp.) Das Plasmid trägt das Gen für β-Lactamase (Ampicillin-Resistenz) aus pBR322 sowie das Gen für alkalische Phosphatase (phoA) aus E.coli K12. Zur Herstellung von pSA302 und pSA506 wurden entsprechende Oligonukleotide in die Erkeπnungssequenzen für die Restriktionsendonukleasεn Ncol bzw. Spl, die sich innerhalb des phoA-Gens befinden, durch DNSKlonierung eingefügt. pHS4133 ist anschließend aus pSA506 durch Entfernung eines Pstl-Fragmentes von 27 Bp hergestellt worden. In den Abb. 2-4 sind Restriktioπskarten der drei neukombinierten Plasmide dargestellt:Plasmid DNA was isolated from the transformants obtained (ampicillin-resistant cells) and the structure of the plasmid was confirmed by restriction analysis. Fig. 1 shows a restriction map of pSB94. (The distances between the individual restriction sequences are given in base pairs (bp), the total size is 6454 bp.) The plasmid carries the gene for β-lactamase (ampicillin resistance) from pBR322 and the gene for alkaline phosphatase (phoA) from E. coli K12. For the production of pSA302 and pSA506, corresponding oligonucleotides were inserted into the recognition sequences for the restriction endonucleases Ncol or Spl, which are located within the phoA gene, by DNA cloning. pHS4133 was then prepared from pSA506 by removing a 27 bp PstI fragment. Fig. 2-4 show restriction maps of the three recombined plasmids:
zu Abb. 2: Die Zahlen 297 und 298 über der Aminosäureteilsequenz kennzeichnen die Aminosäuren der Prä-Phosphatase, zwischen welche in dem Protein SA302/1 die zusätzlichen 6 Aminosäuren eingefügt sind.to Fig. 2: The numbers 297 and 298 above the partial amino acid sequence indicate the amino acids of the pre-phosphatase, between which the additional 6 amino acids are inserted in the protein SA302 / 1.
zu Abb. 3: Die Zahlen 447 und 448 über der Aminosäureteilsequenz kennzeichnen die Aminosäuren der Prä-Phosphatas'e, zwischen welche in dem Protein SA 506/1 die zusätzlichen 15 Aminosäuren eingefügt sind.3: The numbers 447 and 448 above the partial amino acid sequence indicate the amino acids of the pre-phosphatase, between which the additional 15 amino acids are inserted in the protein SA 506/1.
zu Abb. 4: Die Zahlen 447 und 448 über der Aminosäureteilsequenz kennzeichnen die Aminosäuren der Prä-Phosphatase, zwischen welche in dem Protein HS4133/1 die zusätzlichen 6 Aminosäuren eingefügt sind.to Fig. 4: The numbers 447 and 448 above the partial amino acid sequence identify the amino acids of the pre-phosphatase, between which the additional 6 amino acids are inserted in the protein HS4133 / 1.
Bei der Herstellung der neukombinierten Plasmide wurden literaturbekannte Methoden der DNS-Klonierung angewendet. Diese sind von T. Maniatis et al. (Molecular Cloniπg. Cold Spring Harbor Laboratσry, Cold Spring Harbor, N.Y., USA [1982]) ausführlich und zusammenfassend beschrieben worden. Als Wirtszelle, dient der Stamm E.Coli SB 44 (W. Boidol, Dissertation, Technische Universität Berlin [1984]; G. Siewert et al., US-Patent Nr. 4 375 514 [1983]). Dies ist eine phosphatasenegative Mutante (phoA) des weit verbreiteten Klonierungsstammes E.coli HB 101 (hinterlegt am 16.7.79 unter der Nr. DSM 1807). Oligonukleotide wurden mit Hilfe eines automatisierten DNS-Synthesegerätes, Modell 381A der Firma 'Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif. 94 404, USA', nach den Angaben des Geräteherstellers synthetisiert und durch Gelelektrophorese gereinigt.Methods of DNA cloning known from the literature were used in the preparation of the recombined plasmids. These are from T. Maniatis et al. (Molecular Cloniπg. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA [1982]) have been described in detail and in summary. The E.Coli SB 44 strain (W. Boidol, dissertation, Technical University Berlin [1984]; G. Siewert et al., US Pat. No. 4,375,514 [1983]) serves as the host cell. This is a phosphatase negative mutant (phoA) of the widely used cloning strain E. coli HB 101 (deposited on July 16, 1979 under the number DSM 1807). Oligonucleotides were analyzed using an automated model 381A DNA synthesizer from 'Applied Biosystems, 850 Lincoln Center Drive, Foster City, Calif. 94 404, USA ', synthesized according to the manufacturer's instructions and purified by gel electrophoresis.
Die Sequenzierung von DNS erfolgte nach dem Didesoxy-Verfahren (F. Sanger et al., Proc.Natl. Acad. Sei. USA 74 , 5463-5467 [1977]) unter Verwendung der Einzelstrang-Phagenvektoren M13mp18 und M13mp19 (C. Yanisch-Perron et al., Gene 33, 103-119 [1985]), die bei Boehringer (Mannheim), Pharmacia-LKB GmbH und vielen anderen Anbietern erhältlich sind.DNA was sequenced using the dideoxy method (F. Sanger et al., Proc.Natl. Acad. Sci. USA 74, 5463-5467 [1977]) using the single-strand phage vectors M13mp18 and M13mp19 (C. Yanisch- Perron et al., Gene 33, 103-119 [1985]), which are available from Boehringer (Mannheim), Pharmacia-LKB GmbH and many other suppliers.
Zur radioaktiven Markierung wurde Desoxyadenosin-5-[α- 35S]thiotriphosphat eingesetzt (M.D. Biggin et al., Proc. Natl. Acad.Deoxyadenosine 5- [α- 35 S] thiotriphosphate was used for the radioactive labeling (MD Biggin et al., Proc. Natl. Acad.
Sei. USA 80, 3963-3965 [1983]).Be. USA 80, 3963-3965 [1983]).
Die Restriktionsendonukleasen EcoRI, PstI, SphI und Haelll sowie DNS-Ligase (T4) wurden von der Firma Boehringer, Mannheim, bezogen. Ncol war von New England Biolabs GmbH, 6231 Schwalbach, und Polynukleotid-Kinase (T4) von Pharmacia/P-L, 7800 Freiburg i.Br. The restriction endonucleases EcoRI, PstI, SphI and Haelll as well as DNA ligase (T4) were obtained from Boehringer, Mannheim. Ncol was from New England Biolabs GmbH, 6231 Schwalbach, and polynucleotide kinase (T4) from Pharmacia / PL, 7800 Freiburg i.Br.
1.1. Herstellung von pSA 3021.1. Preparation of pSA 302
Das Plasmid pSB94 wurde mit der Restriktionsendonuklease Ncol linearisiert. Dazu wurde eine Reaktionsmischung, welche 14 μg Plasmid-DNS und 17 Einheiten Ncol in 50 μl Reaktionspuffer (50 mM Tris HCl, 10 mM MgCl2, 100 mM NaCl2, 1 mM Dithiothreitol, pH 7.5) enthielt, 7 h bei 37 °C inkubiert. Nach Extraktion der Reaktionsmischung mit Phenol wurde die DNS mit Äthanol gefällt, getrocknet und im TE-Puffer (45 mM Tris HCl, 1 mM EDTA, pH 7.5) gelöst.The plasmid pSB94 was linearized with the restriction endonuclease Ncol. For this purpose, a reaction mixture which contained 14 μg plasmid DNA and 17 units Ncol in 50 μl reaction buffer (50 mM Tris HCl, 10 mM MgCl 2 , 100 mM NaCl 2 , 1 mM dithiothreitol, pH 7.5) was kept at 37 ° C. for 7 hours incubated. After extraction of the reaction mixture with phenol, the DNA was precipitated with ethanol, dried and dissolved in the TE buffer (45 mM Tris HCl, 1 mM EDTA, pH 7.5).
Je 1 μg der beiden 18mer synthetischen Oligonukleotide1 μg each of the two 18mer synthetic oligonucleotides
5'-CAT GGC CCT GCA GGA CCC-3' und 5'-C ATG GGG TCC TGC AGG GC-3', welche an der 5'-OH-Gruppe nicht phosphoryliert waren, wurden in wässriger Lösung gemischt und 15 Min bei 37 °C vorinkubiert. Sie wurden anschließend mit 3.5 μg linearisiertem pSB94 und 4 Einheiten DNS-Ligasse (T4) in 50 μl Ligase-Reaktionspuffer (20 mM Tris HCl, 10 mM MgCl2, 10 mM Dithioerythrit, 0.6 mM ATP, pH 7.6) 2,5 h bei 20 °C umgesetzt. Die DNS wurde wie oben beschrieben durch Phenolextraktion und fithanolfällung aufgearbeitet und zur Transformation des Stammes E.coli SB 44 eingesetzt. Die Herstellung der kompetenten Zellen und die Transformation erfolgten nach einem Verfahren von M. Dagert und S.D. Ehrlich (Gene 6, 23-28 [1979]).5'-CAT GGC CCT GCA GGA CCC-3 'and 5'-C ATG GGG TCC TGC AGG GC-3', which were not phosphorylated on the 5'-OH group, were mixed in aqueous solution and 15 min at 37 ° C pre-incubated. They were then treated with 3.5 μg linearized pSB94 and 4 units of DNA ligasse (T4) in 50 μl ligase reaction buffer (20 mM Tris HCl, 10 mM MgCl 2 , 10 mM dithioerythritol, 0.6 mM ATP, pH 7.6) for 2.5 h 20 ° C implemented. The DNA was processed by phenol extraction and fithanol precipitation as described above and used to transform the strain E. coli SB 44. The competent cells were produced and transformed using a method by M. Dagert and SD Ehrlich (Gene 6, 23-28 [1979]).
Die transformierten Zellen wurden zu Einzelkolonien ausplattiert. Kolonien, deren Plasmid-DNS die o.g. Oligonukleotidse- quenzen enthielten, wurden durch Koloniehybridisierung identifiziert. Die Koloniehybridisierung wurde entsprechend dem von T. Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., USA [1982]) angegebenen Basisprotokolle in einer von J.P. Gergen et al. (Nucleic Acids Research 7 , 2115-2136 [1979]) beschriebenen Variante durchgeführt.The transformed cells were plated out into single colonies. Colonies whose plasmid DNA contains the above-mentioned oligonucleotide sequences were identified by colony hybridization. Colony hybridization was carried out according to the method described by T. Maniatis et al. (Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA [1982]) given basic protocols in a JP Gergen et al. (Nucleic Acids Research 7, 2115-2136 [1979]).
Als radioaktives Hybridisierungsreagenz dienten die o.g. Oligo- nukleotide, die durch Umsetzung mit ɣP 32-ATP und PolynukleotidKinase (T4) zuvor mit 5'-P32-Phosphat markiert worden waren.The above-mentioned oligonucleotides, which had previously been labeled with 5'-P 32 phosphate by reaction with ɣP 32 -ATP and polynucleotide kinase (T4), served as the radioactive hybridization reagent.
In den neukombinierten Plasmiden der positiven Klone wurde die Orientierung des synthetischen Ncol-Fragmentes von 18 Bp durch Restriktionsanalyse ermittelt. Dazu wurde nach Standardverfahren Plasmid-DNS isoliert und mit der Restriktionsendonuklease HaeIII umgesetzt. Die entstandenen Fragmentmischungen wurden durch Gelelektrophorese analysiert (Molekulargewichtsbestimmung durch Vergleich mit HaeIII-geschnittenem pBR322 in einem 8 %igen Polyacrylamidgel).In the newly combined plasmids of the positive clones, the orientation of the synthetic Ncol fragment of 18 bp was determined by restriction analysis. For this purpose, plasmid DNA was isolated according to standard methods and reacted with the restriction endonuclease HaeIII. The resulting fragment mixtures were analyzed by gel electrophoresis (molecular weight determination by comparison with HaeIII-cut pBR322 in an 8% polyacrylamide gel).
Die für die Integration des synthetischen Fragmentes von 18 Bp verwendete Ncol-Schnittstelle ist in pSB94 auf einem HaelllFragment von 385 Bp lokalisiert. Dieses Fragment muß in den neukombinierten Plasmiden fehlen und durch zwei neue Fragmente ersetzt sein, da das 18 Bp-Fragment selbst eine HaeIII-Sequenz trägt. Die Größe der beiden neuen Fragmente hängt von der Orientierung des 18 Bp-Fragmentes ab: in der gewünschten, in Abb.2 dargestellten Orientierung werden Fragmente von 342 Bp und 61 Bp erwartet, während die entgegengesetzte Orientierung Fragmente von 330 Bp und 73 Bp ergibt. Es wurden mehrere Plasmide beider Orientierungen gefunden. Das in Abb. 2 dargestellte pSA302 ist eines der Plasmide mit der gewünschten Orientierung.The Ncol interface used for the integration of the synthetic fragment of 18 bp is located in pSB94 on a Haelll fragment of 385 bp. This fragment must be missing in the recombined plasmids and replaced by two new fragments, since the 18 bp fragment itself carries a HaeIII sequence. The size of the two new fragments depends on the orientation of the 18 bp fragment: in the desired orientation shown in FIG. 2, fragments of 342 bp and 61 bp are expected, while the opposite orientation fragments of 330 bp and 73 bp. Several plasmids of both orientations were found. The pSA302 shown in Fig. 2 is one of the plasmids with the desired orientation.
Das EcoRI-Fragment von 349 Bp aus PSA302, welches die durch Klonierung eingefügte synthetische DNS enthält, wurde in den Sequenziervektor M13mp19 kloniert und, wie oben angegeben, sequenziert. Die in Abb. 2 dargestellte Teilsequenz von pSA302 zwischen den beiden EcoRI-Schnittstellen ließ sich voll bestätigen.The 349 bp EcoRI fragment from PSA302, which contains the synthetic DNA inserted by cloning, was cloned into the sequencing vector M13mp19 and sequenced as indicated above. The partial sequence of pSA302 shown in Fig. 2 between the two EcoRI interfaces was fully confirmed.
1.2. Herstellung von pSA5061.2. Manufacture of pSA506
Die Herstellung erfolgte ähnlich die diejenige von pSA302. pSB94 wurde mit der Restriktionsendonuklease SphI linearisiert; Reaktionsbedingungen und Aufarbeitung waren die gleichen wie für pSA302 beschrieben.The preparation was similar to that of pSA302. pSB94 was linearized with the restriction endonuclease SphI; Reaction conditions and workup were the same as described for pSA302.
Die beiden 45mer OligonukleotideThe two 45mer oligonucleotides
5'-GTCCTGCAGGCCCGGCGGGTCCAGCAGGCCCTGCAGGTCCGCATG-3 ' und 5'-CGGACCTGCAGGGCCTGCTGGACCCGCCGGGCCTGCAGGACCATG-3 ' wurden mit SphI-linearisiertem pSB94 und DNS-Ligase unter den gleichen Bedingungen, wie für pSA302 beschrieben, umgesetzt und aufgearbeitet. Nach Transformation von E.coli SB 44 wurden positive Klone durch Koloniehybridisierung mit den P 32-endmarkierten5'-GTCCTGCAGGCCCGGCGGGTCCAGCAGGCCCTGCAGGTCCGCATG-3 'and 5'-CGGACCTGCAGGGCCTGCTGGACCCGCCGGGCCTGCAGGACCATG-3' were reacted with SphI-linearized pSB94 and DNA ligase for the same conditions as described under the same conditions as described on p30 and DNA ligase pSA2. After transformation of E. coli SB 44, positive clones were labeled with the P 32 end by colony hybridization
45mer Oligonukleotiden identifiziert. Die so erhaltenen neukombinierten Plasmide haben nur auf einer Seite des aus den beiden 45mer-Oligonukleotiden gebildeten DNS- Abschnittes eine intakte Sphl-Sequenz. Diese hat in den gesuchten Plasmiden einen Abstand von 257 Bp von der nächstgelegenen EcoRI-Sequenz (vergl. Abb. 3), während diese Distanz in den Plasmiden der entgegengesetzten Orientierung genau wie in pSB94 nur 212 Bp beträgt.45mer oligonucleotides identified. The recombined plasmids obtained in this way have an intact Sphl sequence only on one side of the DNA section formed from the two 45mer oligonucleotides. In the plasmids sought, this is at a distance of 257 bp from the closest EcoRI sequence (see FIG. 3), while this distance in the plasmids of the opposite orientation is only 212 bp, just as in pSB94.
Die Größenbestimmung der DNS-Fragmente, die durch Umsetzung der neukombinierten Plasmide mit den Restriktionssequenzen EcoRI und SphI erhalten wurden, durch Gelelektrophorese zeigte, daß pSA506 eines der Plasmide mit der gewünschten Orientierung des synthetischen DNS-Fragmentes ist. Zur Bestätigung der in Abb.3 dargestellten Sequenz wurde das EcoRI/Sphl-Fragment von 257 Bp in den Sequenziervektor M13mpl8 kloniert und sequenziert.The size of the DNA fragments obtained by reacting the newly combined plasmids with the restriction sequences EcoRI and SphI by gel electrophoresis showed that pSA506 is one of the plasmids with the desired orientation of the synthetic DNA fragment. To confirm the sequence shown in Fig.3, the EcoRI / Sphl fragment of 257 bp was cloned into the sequencing vector M13mpl8 and sequenced.
1.3. Herstellung von pHS41331.3. Preparation of pHS4133
Eine Reaktionsmischung aus 3 μg DNS des Plasmids pSA506 und 5 Einheiten des Restriktionsenzyms PstI in 50 μl Reaktionspuffer (wie in 1.1.) wurde 2 h bei 37 °C inkubiert und wie in Beispiel 1.1. aufgearbeitet. Es entstanden 3 DNS-Fragmente (27 Bp, 2420 Bp und 4007 Bp), die durch Elektrophorese in einem 1 %igen Agarosegel voneinander getrennt wurden. Die beiden größeren Fragmente wurden durch Elektroelution aus dem Gel isoliert, vereinigt und mit DNS-Ligase (T4) unter Reaktionsbedingungen wie in Beispiel 1.1. umgesetzt, aufgearbeitet und zur Transformation von E.coli SB 44 eingesetzt. Aus einigen Ampicillin-resistenten Klonen wurde Plasmid-DNS isoliert. Eines von diesen ist pHS4133, dessen Restriktionskarte in Abb. 4 dargestellt ist. pHS4133 leitet sich aus pSA506 durch eine Deletion ab, die zu einer Größenverminderung des EcoRI/Sphl-Fragmentes von 257 auf 230 Bp führt. Dieses Fragment wurde wie in Beispiel 1.2. in M13mp18 kloniert und sequenziert. Dabei ließ sich die in Abb. 4 angegebene Nukleotidsequenz bestätigen.A reaction mixture of 3 μg DNA of the plasmid pSA506 and 5 units of the restriction enzyme PstI in 50 μl reaction buffer (as in 1.1.) Was incubated for 2 h at 37 ° C. and as in Example 1.1. worked up. 3 DNA fragments (27 bp, 2420 bp and 4007 bp) were obtained, which were separated from one another by electrophoresis in a 1% agarose gel. The two larger fragments were isolated from the gel by electroelution, combined and with DNA ligase (T4) under the reaction conditions as in Example 1.1. implemented, processed and for transformation used by E.coli SB 44. Plasmid DNA was isolated from some ampicillin-resistant clones. One of these is pHS4133, the restriction map of which is shown in Fig. 4. pHS4133 is derived from pSA506 by a deletion which leads to a reduction in the size of the EcoRI / Sphl fragment from 257 to 230 bp. This fragment was as in Example 1.2. cloned and sequenced in M13mp18. The nucleotide sequence shown in Fig. 4 was confirmed.
2. Herstellung der Proteine SA 302/1, SA 506/1 und HS 4133/12. Production of the proteins SA 302/1, SA 506/1 and HS 4133/1
Die drei Proteine wurden durch Fermentation der in Teil 1 der Beispiele beschriebenen StämmeThe three proteins were obtained by fermentation of the strains described in part 1 of the examples
E.coli SB44 (pSA302),E.coli SB44 (pSA302),
E.coli SB44 (pSA506) und E.coli SB44 (pHS4133) erhalten. Ihre Biosynthese ist in der gleichen Weise wie diejenige von alkalischer Phosphatase reguliert: sie wird durch anorganisches Phosphat reprimiert und ist umgekehrt in phosphatarmen Medien induziert (A. Torriani, Biochem. Biophys. Acta 38, 460-469 [1960]).E.coli SB44 (pSA506) and E.coli SB44 (pHS4133) obtained. Their biosynthesis is regulated in the same way as that of alkaline phosphatase: it is repressed by inorganic phosphate and, conversely, is induced in low-phosphate media (A. Torriani, Biochem. Biophys. Acta 38, 460-469 [1960]).
Zur Identifizierung der Phosphatase-Derivate wurde u.a. das Immunblot-Verfahren nach H. Towbin et al. (Proc. Natl. Acad. Sei. USA 76, 4350-4354 [1979]) angewendet. Das dafür benötigte Antiserum war durch Immunisierung von Kaninchen mit gereinigter alkalischer Phosphatase aus E.coli K12 unter Standardbedingungen (vergl. D.M. Weir, Handbook of Experimental Immunology, Vol. III, Blackwell Scientific Publ, Oxford 1978) hergestellt worden. Die zur Immunisierung eingesetzte Phosphatase wurde nach bekannten Verfahren (A. Torriani, in: 'Methods in Enzymology', Vol. XIIB, 212-218, L. Grossman, K. Moldave (Eds.) Academic Press, New York [1968]) isoliert. Als. zweiter Antikörper diente ein Ziegen-Antiserum gegen Kaninchen-IgG, an welches alkalische Phosphatase als Marker-Enzym gekoppelt war (Cooper Biomedical Inc., Malvern, USA). 5-Brom-4-chlor-3-indolylphosphat wurde als Farbreagenz verwendet.To identify the phosphatase derivatives, the immunoblot method according to H. Towbin et al. (Proc. Natl. Acad. Sci. USA 76, 4350-4354 [1979]). The antiserum required for this was obtained by immunizing rabbits with purified alkaline phosphatase from E. coli K12 under standard conditions (see DM Weir, Handbook of Experimental Immunology, Vol. III, Blackwell Scientific Publ, Oxford 1978). The phosphatase used for the immunization was obtained by known methods (A. Torriani, in: 'Methods in Enzymology', Vol. XIIB, 212-218, L. Grossman, K. Moldave (Eds.) Academic Press, New York [1968]) isolated. As. the second antibody was a goat antiserum against rabbit IgG, to which alkaline phosphatase was coupled as a marker enzyme (Cooper Biomedical Inc., Malvern, USA). 5-bromo-4-chloro-3-indolyl phosphate was used as a color reagent.
Die drei Proteine sowie auch die drei o.g. Stämme, von denen sie produziert werden, sind untereinander so ähnlich, daß für alle das gleiche Fermentations- und Isolierungsverfahren angewendet werden kann.The three proteins as well as the three above Strains from which they are produced are so similar to one another that the same fermentation and isolation process can be used for all of them.
2.1. Fermentation2.1. fermentation
20 ml L-Medium (E.S. Lennox, Virology 1, 190-206 [1955]), die zusätzlich 100 μg/ml Ampicillin enthielten, wurden mit einer Einzelkolonie beimpft, 24 h bei 37 °C geschüttelt und anschließend in 800 ml des gleichen Mediums überführt, die wiederum 16 h bei 37 °C geschüttelt wurden. Diese Vorzueht wurde in einem 20 1-Glasfermenter überführt, in dem sich 9,2 1 Niedrigphosphatmedium befanden (LP-Medium nach K. Kreuzer et al., Geneties 81, 459-468 [1975]). Der Fermenter wurde 6 h bei 37 °C und einer Belüftung von 10 1/min mit 220 min-1 gerührt. Anschließend wurden die Zellen in einer Durchflußzentrifuge geerntet, wobei ca. 30 g feuchte Zellmasse erhalten wurde. 2.2. Isolierung von angereicherten Proteinextrakten20 ml L medium (ES Lennox, Virology 1, 190-206 [1955]), which additionally contained 100 μg / ml ampicillin, were inoculated with a single colony, shaken at 37 ° C. for 24 h and then in 800 ml of the same medium transferred, which in turn were shaken at 37 ° C for 16 h. This procedure was transferred to a 20 l glass fermenter containing 9.2 l of low phosphate medium (LP medium according to K. Kreuzer et al., Geneties 81, 459-468 [1975]). The fermenter was stirred for 6 h at 37 ° C. and aeration at 10 1 / min at 220 min -1 . The cells were then harvested in a flow-through centrifuge, about 30 g of moist cell mass being obtained. 2.2. Isolation of enriched protein extracts
30 g feuchte Zellen aus 2.1. wurden in 400 ml Aufschlußpuffer (50 mM Tris HCl, 160 mM NaCl, pH 8.0) suspendiert. Nach Zugabe von Phenylmethylsulfonylfluorid (Endkonzentration 10 mM) und DNAse (DNAse I aus Rinderpankreas, Fa. Boehringer/Mannheim, Reinheitsgrad I; 10 μg/g feuchte Zellen) wurde die Suspension in einem Zeilhomogenisator (Manton-Gaulin) bei einem Druck von 650-700 kg/cm2 (10 000 Psi) in einem Kühlraum (4 °C) aufgeschlossen. Der Aufschluß wurde zweimal wiederholt, nach jedem Durchgang wurde die Suspension in Eis gekühlt. Die unlöslichen Zellbestandteile wurden durch Zentrifugation abgetrennt (20 min. bei 10 000 × g und 4 °C), zweimal mit Aufschlußpuffer gewaschen und in 50 mM Tris HCl, pH 8.0/8 M Harnstoff (60 ml) gelöst. Die Lösung wurde durch Zentrifugation von unlöslichen Bestandteilen befreit und bis zur weiteren Reinigung durch Gradientenchromatographie in Eis aufbewahrt.30 g moist cells from 2.1. were suspended in 400 ml digestion buffer (50 mM Tris HCl, 160 mM NaCl, pH 8.0). After adding phenylmethylsulfonyl fluoride (final concentration 10 mM) and DNAse (DNAse I from bovine pancreas, Boehringer / Mannheim, purity grade I; 10 μg / g moist cells), the suspension was placed in a cell homogenizer (Manton-Gaulin) at a pressure of 650- 700 kg / cm 2 (10,000 psi) digested in a cold room (4 ° C). The digestion was repeated twice, after each run the suspension was cooled in ice. The insoluble cell components were separated by centrifugation (20 min. At 10,000 × g and 4 ° C.), washed twice with digestion buffer and dissolved in 50 mM Tris HCl, pH 8.0 / 8 M urea (60 ml). The solution was freed from insoluble constituents by centrifugation and kept in ice until further purification by gradient chromatography.
2.3. Identifizierung der Phosphatase-Derivate2.3. Identification of the phosphatase derivatives
Hauptproteinbestandteil (ca. 50-70 %) der in 2.2. erhaltenen Proteinextrakte in Harnstoffpuffer ist das jeweilige Phospha- tase-Derivat. Nach Trennung der Prσteinmischungen durch Elektrophorese in SDS-Polyacrylamidgelen unter Standardbedingungen (U.K. Laemmli, Nature 227, 680-685 [1970]) wurden die entsprechenden Proteinbanden anhand folgender Kriterien identifiziert: a) Die stärkste Bande des Proteingemisches hat ein Molekulargewicht, welches der in Tab. 1 angegebenen Aminosäuresequenz entspricht. b) Dieses Protein ist durch Phosphat reprimierbar, d.h. die Bande fehlt, wenn die Zellen wie in 2.1., jedoch mit der 10-fachen Phosphatkonzentration fermentiert wurden. c) Die Bande ist plasmidspezifisch, sie fehlt in Zellen des plasmidfreien Stammes E.coli SB 44, der unter den gleichen Bedingungen wie in 2.1. hergestellt wurde. d) Die Bande reagiert im Immunblot spezifisch mit Antiserum gegen gereinigte alkalische Phosphatase aus E.coli K12.Main protein component (approx. 50-70%) of the 2.2. The protein extract obtained in urea buffer is the respective phosphatase derivative. After separation of the protein mixtures by electrophoresis in SDS polyacrylamide gels under standard conditions (UK Laemmli, Nature 227, 680-685 [1970]), the corresponding protein bands were identified using the following criteria: a) The strongest band of the protein mixture has a molecular weight which corresponds to the amino acid sequence given in Table 1. b) This protein can be repressed by phosphate, ie the band is missing if the cells have been fermented as in 2.1., but at 10 times the phosphate concentration. c) The band is plasmid-specific, it is absent in cells of the plasmid-free strain E. coli SB 44, which under the same conditions as in 2.1. was produced. d) The band reacts specifically in the immunoblot with antiserum against purified alkaline phosphatase from E. coli K12.
Aus gelelektrophoretischen Molekulargewichtsbestimmungen mit Hilfe eines Standardproteingemisches (Low Molcular Weight Proteins der Firma Pharmacia) ist zu erkennen, daß die PhosphataseDerivate in der sogen. Prä-Form vorliegen, d.h. noch die Signalsequenz der alkalischen Phosphatase enthalten. Die Proteine sind in verdünntem Puffer relativ schwer löslich. Daher lassen sich in den nach. Zellaufschluß und Zentrifugation erhaltenen Überstände nur geringe Mengen nachweisen.From gel electrophoretic molecular weight determinations with the aid of a standard protein mixture (low molecular weight protein from Pharmacia) it can be seen that the phosphatase derivatives in the so-called. Pre-form, i.e. still contain the signal sequence of alkaline phosphatase. The proteins are relatively sparingly soluble in dilute buffer. Therefore, in the. Detect supernatants obtained from cell disruption and centrifugation only in small amounts.
2.4. Reinigung der Phosphatase-Derivate2.4. Purification of the phosphatase derivatives
Die in 2.2. erhaltenen Proteinextrakte wurden zur weiteren Reinigung der modifizierten Phosphatasen durch Gradientenchromatographie mit Hilfe eines Mitteldruck-Chromatographiegerätes (FPLC) der Fa. Pharmacia, Uppsala/Schweden, aufgetrennt. Eine Anionenaustauschersäule des gleichen Herstellers (Typ Mono Q-HR 5/5) wurde mit Puffer A (50 mM Tris HCl, 8 M Harnstoff, pH 8.0) equilibriert, mit der Proteinlösung aus 2.1. beladen und schließlich mit einem aus Puffer A und Puffer B (50 mM Tris HCl, 1 M NaCl, 8 M Harnstoff, pH 8.0) gebildeten Gradienten von 0-200 mM NaCl mit einer Durchflußrate von 1.0 ml/min eluiert. Das gesamte Gradientenvolumen betrug 20 ml, es wurden Fraktionen von je 1 ml gesammelt.The in 2.2. Protein extracts obtained were separated for further purification of the modified phosphatases by gradient chromatography with the aid of a medium pressure chromatography device (FPLC) from Pharmacia, Uppsala / Sweden. An anion exchange column from the same manufacturer (type Mono Q-HR 5/5) was equilibrated with buffer A (50 mM Tris HCl, 8 M urea, pH 8.0), with the protein solution from 2.1. loaded and finally eluted with a gradient of 0-200 mM NaCl formed from buffer A and buffer B (50 mM Tris HCl, 1 M NaCl, 8 M urea, pH 8.0) at a flow rate of 1.0 ml / min. The total gradient volume was 20 ml, fractions of 1 ml were collected.
Die Extinktion des Eluats bei 280 nm wurde mit einem Durchflußplotometer registriert. Die Proteinzusammensetzung der einzelnen Fraktionen wurde durch SDS-Polyacrylamid-Gelelektrophorese analysiert. Die Phosphatase-Derivate eluierten bei 50-70 mM NaCl. Die entsprechenden Fraktionen wurden vereinigt und gegen das 400-fache Volumen Puffer (50 mM Tris HCl, 150 mM NaCl, pH 8.0) dialysiert. Die Proteine blieben unter diesen Bedingungen gelöst und wurden in dieser Form für die anschließende Spaltung mit Kollagenase eingesetzt. Die Reinheit der Präparationen wurce durch SDS-Polyacrylamid-Gelelektrophorese kontrolliert (siehe S. 35 , Spur 4, 6 und 8), sie war größer als 90 %. Der Proteingehalt wurde nach W. Schaffner et al. (Anal. Biochem. 56, 502-514 [1973]) mit Amidoschwarz 10B bestimmt, die Konzentrationen betrugen 0.6-0.7 mg/ml. Aus einer 10 1-Fermentation wurden ca. 160 mg Protein erhalten. 3. Spezifische Spaltung der modifizierten Phosphatase-Proteine mit KollagenaseThe absorbance of the eluate at 280 nm was recorded with a flow plotometer. The protein composition of the individual fractions was analyzed by SDS-polyacrylamide gel electrophoresis. The phosphatase derivatives eluted at 50-70 mM NaCl. The appropriate fractions were combined and dialyzed against 400 times the volume of buffer (50 mM Tris HCl, 150 mM NaCl, pH 8.0). The proteins remained dissolved under these conditions and were used in this form for the subsequent cleavage with collagenase. The purity of the preparations was checked by SDS-polyacrylamide gel electrophoresis (see p. 35, lanes 4, 6 and 8), it was greater than 90%. The protein content was determined according to W. Schaffner et al. (Anal. Biochem. 56, 502-514 [1973]) with amido black 10B, the concentrations were 0.6-0.7 mg / ml. Approx. 160 mg protein was obtained from a 10 l fermentation. 3. Specific cleavage of the modified phosphatase proteins with collagenase
3.1. Reinigung der Kollagenase3.1. Purification of the collagenase
Clostridiopeptidase A aus Clostridium histolyticum (EC 3.4.24.3) wurde von der Fa. Sigma Chemie GmbH, D-8024 Deisenhofen, Grünwalder Weg 30, bezogen (Typ VII, Best. -Nr. C0773). Das Enzym wurde durch Gradientenchromatographie unter Verwendung des FPLC-Gerätes und der gleichen Anionenaustauschersäule wie in Beispiel 2.4. weiter gereinigt. Es wurden 2 mg Kollagenase eingesetzt und mit einem aus Puffer A (10 Tris HCl, 5 mM CaCl2, pH 8.0) und Puffer B (10 mM Tris HCl, 5 mM CaCl2, 1 mM NaCl, pH 8.0) gebildeten Gradienten von 0-200 mM NaCl mit einer Durchflußgeschwindigkeit von 0.5 ml/min eluiert. Das Gesamtvolumen des Gradienten betrug 20 ml. Die Kollagenase-Aktivität wurde mit einem von E. Wünsch et al. (Hoppe-Seyler's Z. Physiol. Chem. 333, 149-151 [1963]) beschriebenen Test ermittelt. Die aktivste Fraktion hatte einen Proteingehalt von 0.8 mg/ml und eine spezifische Aktivität von 2500 Einheiten/mg. Die im Folgenden beschriebenen Spaltversuche wurden mit dieser Fraktion ausgeführt, die bei -20 °C aufbewahrt wurde. 3.2. Spaltung von HS 4133/1Clostridiopeptidase A from Clostridium histolyticum (EC 3.4.24.3) was obtained from Sigma Chemie GmbH, D-8024 Deisenhofen, Grünwalder Weg 30 (Type VII, order no. C0773). The enzyme was purified by gradient chromatography using the FPLC and the same anion exchange column as in Example 2.4. further cleaned. 2 mg of collagenase were used and with a gradient of 0 formed from buffer A (10 Tris HCl, 5 mM CaCl 2 , pH 8.0) and buffer B (10 mM Tris HCl, 5 mM CaCl 2 , 1 mM NaCl, pH 8.0) -200 mM NaCl eluted at a flow rate of 0.5 ml / min. The total volume of the gradient was 20 ml. The collagenase activity was determined using one of E. Wünsch et al. (Hoppe-Seyler's Z. Physiol. Chem. 333, 149-151 [1963]). The most active fraction had a protein content of 0.8 mg / ml and a specific activity of 2500 units / mg. The cleavage tests described below were carried out with this fraction, which was kept at -20 ° C. 3.2. Cleavage of HS 4133/1
Das einkettige Protein hat eine Länge von 477 Aminosäuren (Molekulargewicht 49 919). Es enthält die durch Kollagenasen spaltbare SequenzThe single-chain protein has a length of 477 amino acids (molecular weight 49,919). It contains the sequence that can be cleaved by collagenases
-Gly-(Pro-Y-Gly)2-Pro- wobei für Y einmal His und einmal Ala steht (vergl. Tab. 1). Durch Spaltung beider Y-Gly-Bindungen entstehen zwei Proteine mit Längen von 447 Aminosäuren (MG. 46 880) und 27 Aminosäuren (MG. 2832) sowie das Tripeptid Gly-Pro-Ala. Um die Reaktionsfähigkeit der verschiedenen Phosphatase-Derivate gegenüber Kollagenase miteinander vergleichen zu können, wurde die Bildung des größten Spaltproduktes in Abhängigkeit von der Reaktionszeit und der Enzymmenge gemessen.-Gly- (Pro-Y-Gly) 2 -Pro- where Y is His and Ala (see Table 1). By cleaving both Y-Gly bonds, two proteins with lengths of 447 amino acids (MW. 46 880) and 27 amino acids (MG. 2832) as well as the tripeptide Gly-Pro-Ala are formed. In order to be able to compare the reactivity of the various phosphatase derivatives with collagenase, the formation of the largest cleavage product was measured as a function of the reaction time and the amount of enzyme.
Eine Reaktionsmischung, welche 53 μg HS 4133/1 und 0.1 Einheit Clostridiopeptidase A in 100 μl Reaktionspuffer (90 mM Tris HCl, 5 mM CaCl2, 120 mM NaCl, pH 8.0) enthielt, wurde 2,5 h bei 37 °C inkubiert. In Zeitabständen von 15 bis 30 Min. wurden Proben von je 10 μl entnommen und durch Zugabe von 3 μl Probenpuffer (10 % Saccharose, 10 % Natriumdodeeylsulfat, 1 mM Dithiothreitol, 2 mM EDTA) sowie durch fünfminütiges Erhitzen auf 95 °C sofort gestoppt.A reaction mixture which contained 53 μg HS 4133/1 and 0.1 unit clostridiopeptidase A in 100 μl reaction buffer (90 mM Tris HCl, 5 mM CaCl 2 , 120 mM NaCl, pH 8.0) was incubated at 37 ° C. for 2.5 h. Samples of 10 μl each were taken at intervals of 15 to 30 minutes and stopped immediately by adding 3 μl of sample buffer (10% sucrose, 10% sodium dodeeyl sulfate, 1 mM dithiothreitol, 2 mM EDTA) and heating to 95 ° C. for five minutes.
Die Proben wurden durch Elektrophorese in einem SDS-Polyacrylamid-Gradientengel (Gradient von 12,5 % bis 25 % Acrylamid mit konstant 0,33 % Bisacrylamid) getrennt. Nach Anfärben mit Coomassie Brilliant Blue G wurde das Gel entsprechend den Protein bahnen in Streifen geschnitten und mit einem Gel-Scanner (Fa . Isco , optische Einheit Typ 6 , Absorptionsmonitor UA-5 ) bei 280 nm quantitativ ausgewertet. Mit Hilfe einer entsprechenden Eichkurve , die mit einem gereinigten Phosphatase-Protein erhalten worden war , wurden die Proteingehalte für die Banden des größten Spaltproduktes aus den Peakhöhen ermittelt. Die Ergebnisse sind auf S. .36 zusammengefaßt . Aus den Daten ergibt sich eine Produktbildungsrate von 21 .0 pMol/min/Enzymeinheit für HS 4133 /1.The samples were separated by electrophoresis in an SDS-polyacrylamide gradient gel (gradient from 12.5% to 25% acrylamide with a constant 0.33% bisacrylamide). After staining with Coomassie Brilliant Blue G, the gel became the protein strips cut into strips and evaluated quantitatively at 280 nm using a gel scanner (Isco, optical unit type 6, absorption monitor UA-5). With the help of a corresponding calibration curve, which had been obtained with a purified phosphatase protein, the protein contents for the bands of the largest cleavage product were determined from the peak heights. The results are summarized on p. .36. The product results in a product formation rate of 21 .0 pmol / min / enzyme unit for HS 4133/1.
Die Trennung auf S . 35 zeigt in Spur 7 eine gelelektrophoretisehe Trennung des größten Spaltproduktes aus einer Probe von HS 4133 /1 nach vollständiger Umsetzung mit Clostridiopeptidase A.The separation on p. 35 shows in lane 7 a gel electrophoresis separation of the largest cleavage product from a sample of HS 4133/1 after complete reaction with clostridiopeptidase A.
3 .3 . Spaltung von SA 506 /13 .3. Splitting of SA 506/1
Das einkettige Protein hat eine Länge von 486 Aminosäuren (MG .The single-chain protein has a length of 486 amino acids (MG.
50 596 ) und enthält die durch Kollagenase spaltbare Sequenz wobei für Y einmal His und viermal Ala steht (vergl. Tab. 1). Bei Spaltung aller Y-Gly-Bindungen entstehen die gleichen Proteine wie aus HS 4133/1 (vergl. 3.2.) und 4 Mol Gly-Pro-Ala je Mol Substrat. Die Bestimmung der Reaktionsfähigkeit von SA 506/1 gegenüber Clostridiopeptidase A erfolgte in der gleichen Weise wie bei HS 4133/1.50 596) and contains the sequence cleavable by collagenase where Y stands for His and four times Ala (see Table 1). When all Y-Gly bonds are cleaved, the same proteins are produced as from HS 4133/1 (cf. 3.2.) And 4 moles of Gly-Pro-Ala per mole of substrate. The reactivity of SA 506/1 towards clostridiopeptidase A was determined in the same way as for HS 4133/1.
Der Reaktionsansatz enthielt 53 μg S 506/1 in 100 μl und hatte die gleichen Enzym- und Pufferkonzentrationen wie der Ansatz für HS 4133 / 1 . Er wurde 80 Min . bei 37 °C inkubiert. In Abständen von 10 bis 15 Min . wurden 10 μl-Proben genommen und , wie in 3 . 2 . beschrieben, gestoppt und nach Gelelektrophorese mit einem GelScanner analysiert . Die Ergebnisse sind auf s. 36 dargestellt. Die Produktbildungsrate betrug 60 p Mol /min/Enzymeinheit.The reaction mixture contained 53 μg S 506/1 in 100 μl and had the same enzyme and buffer concentrations as the mixture for HS 4133/1. He was 80 min. incubated at 37 ° C. Every 10 to 15 minutes 10 ul samples were taken and, as in 3. 2nd described, stopped and analyzed after gel electrophoresis with a gel scanner. The results are on p. 36 shown. The product formation rate was 60 p mol / min / enzyme unit.
Die Trennung auf S. 35 zeigt in Spur 5 das größte Reaktionsprodukt aus SA 506 /1 nach quantitativer Umsetzung mit Clostridiopeptidase A.The separation on p. 35 shows in lane 5 the largest reaction product from SA 506/1 after quantitative conversion with clostridiopeptidase A.
3 . 4 . Spaltung von SA 302/13rd 4th Splitting of SA 302/1
Das einkettige Protein hat eine Länge von 477 Aminosäuren und enthält die durch Kollagenase spaltbare Sequenz The single-chain protein is 477 amino acids in length and contains the sequence that can be cleaved by collagenase
(vergl. Tab. 1). Es entstehen zwei Spaltprodukte mit Längen von(see Tab. 1). Two fission products with lengths of
301 Aminosäuren (MG. 31 269) und 176 Aminosäuren (MG. 18 668).301 amino acids (MW. 31,269) and 176 amino acids (MG. 18,668).
Wegen der geringen Reaktionsfähigkeit von SA 302/1 wurde nur ein ungefährer Wert für die Produktbildungsrate ermittelt.Due to the low reactivity of SA 302/1, only an approximate value for the product formation rate was determined.
Eine Reaktionsmischung, welche 5 μg SA 302/1 und 1,0 Einheit Clostridiopeptidase A in 15 μl Reaktionspuffer (wie in 3.2.) enthielt, wurde 15 h bei 37 °C inkubiert und dann, wie in 3.2. beschrieben, durch Zugabe von Probenpuffer und Erhitzen gestoppt und durch Gelelektrophorese und Auswertung mit einem Gel-Scanner analysiert. Es waren ca.0,02 p Mol/min/Enzymeinheit des größeren Reaktionsproduktes gebildet worden. Auf Seite 35 ist in Spur 9 die entsprechende gelelektrophoretisehe Trennung dargestellt. Tab. 1: Aminosäure-Sequenzen der von alkalischer Phosphatase (AP) abgeleiteten Modell- und FusionsproteineA reaction mixture which contained 5 μg SA 302/1 and 1.0 unit of clostridiopeptidase A in 15 μl reaction buffer (as in 3.2.) Was incubated at 37 ° C. for 15 h and then, as in 3.2. described, stopped by adding sample buffer and heating and analyzed by gel electrophoresis and evaluation with a gel scanner. About 0.02 p mol / min / enzyme unit of the larger reaction product had been formed. The corresponding gel electrophoresis separation is shown on page 35 in lane 9. Tab. 1: Amino acid sequences of the model and fusion proteins derived from alkaline phosphatase (AP)
ProteinbezeichnungProtein name
SA506/1 Phos(1-444)-Gly-Pro-His-Gly-(Pro-Ala-Gly)4-Pro-His-Phos(448-471) (486 AS) HS4133/1 Phos(1-444)-Gly-Pro-His-Gly-Pro-Ala-Gly-Pro-His-Phos(448-471) (477 AS) SA302/1 Phos(1-297)-His-Gly-Pro-Ala-Gly-Pro-His-Gly-Phos(300-471) (477 AS) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -SA506 / 1 Phos (1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly) 4 -Pro-His-Phos (448-471) (486 AS) HS4133 / 1 Phos (1-444) -Gly-Pro-His-Gly-Pro-Ala-Gly-Pro-His-Phos (448-471) (477 AS) SA302 / 1 Phos (1-297) -His-Gly-Pro-Ala-Gly-Pro -His-Gly-Phos (300-471) (477 AS) - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -
SA341/1 Phos (1-444)-Gly-Pro-His-Gly-(Pro-Ala-Gly)4-Pro-ACTH(1-39) (500 AS)SA341 / 1 Phos (1-444) -Gly-Pro-His-Gly- (Pro-Ala-Gly) 4 -Pro-ACTH (1-39) (500 AS)
Die Phosphatasesequenzen auf beiden Seiten der Collagenase-Schnittstellen sind durch die Abkürzung 'Phos' sowie die in Klammern gesetzten Positionsnummern der ersten und letzten Aminosäure der jeweiligen Teilsequenz gekennzeichnet, wobei sich die Numerierung auf die native Prä-Phosphatase (471 Aminosäuren) aus E.coli K12 bezieht. Die Sequenz für ACTH (1-39) ist in der deutschen Patentanmeldung (Aktenzeichen P37 31 875.6 )dargestellt. The phosphatase sequences on both sides of the collagenase interfaces are characterized by the abbreviation 'Phos' and the position numbers in parentheses of the first and last amino acid of the respective partial sequence, the numbering being based on the native pre-phosphatase (471 amino acids) from E. coli K12 relates. The sequence for ACTH (1-39) is shown in the German patent application (file number P37 31 875.6).
Tab . 2 : Produktbildungsraten für die Umsetzung der modifizierten Prä-Phosphatasen (Tab . 1 ) und der Prä-Phosphatase/ ATCH-Fusionsproteine (s. Aktenzeichen P 3731 875.6 ) mit Clostridiopeptidase ATab. 2: Product formation rates for the implementation of the modified pre-phosphatases (Tab. 1) and the pre-phosphatase / ATCH fusion proteins (see file number P 3731 875.6) with clostridiopeptidase A
ProteinZahl der repetitiven Produktbildungsbezeichnung Collagenase- rateProtein number of the repetitive product formation name collagenase rate
Schnittstellen pMol/min/Enzymn einheitInterfaces pMol / min / Enzymn unit
SA302/1 1 ~0,02SA302 / 1 1 ~ 0.02
HS4133/1 2 21,0HS4133 / 1 2 21.0
SA506/1 5 59,8SA506 / 1 5 59.8
SA186/1 1 < 0,01SA186 / 1 1 <0.01
SA343/1 2 40SA343 / 1 2 40
SA341/1 5 350SA341 / 1 5 350
SA360/1 8 540 SA360 / 1 8 540
Abb. 5: Gelelektrophoretisehe Trennung der Phosphatase-Proteine aus Tabelle 1 sowie der daraus durch Umsetzung mit Clostridiopeptidase A erhaltenen ReaktionsprodukteFig. 5: Gel electrophoresis separation of the phosphatase proteins from Table 1 and the reaction products obtained therefrom by reaction with Clostridiopeptidase A.
Reihe 1 und 10 Molekulargewichtsmarke Reihe 2, 4, 6 und 8 Proteine aus Tabelle 1 Reihe 3, 5, 7 und 9 Produkte nach Spaltung mit Clostridiopeptidase ARow 1 and 10 molecular weight mark row 2, 4, 6 and 8 proteins from Table 1 row 3, 5, 7 and 9 products after clostridiopeptidase A cleavage
Das in Reihe 2 und 3 eingesetzte Fusionsprotein war nicht durch Gradientenchromatographie gereinigt worden.The fusion protein used in rows 2 and 3 had not been purified by gradient chromatography.
Die Bande mit dem niedrigsten Molekulargewicht in Reihe 3 ist Gly-Pro-ACTH (1-39).The lowest molecular weight band in row 3 is Gly-Pro-ACTH (1-39).
In Reihe 5 und 7 ist nur das hochmolekulare Spaltprodukt zu erkennen, das niedermolekulare Produkt hat das Gel vollständig durchlaufen. In Reihe 9 sind außer den Produkten der Spaltung mit Clostridiopeptidase A (ME 31 200 und 18 700) auch Produkte unspezifischer Spaltung enthalten.In rows 5 and 7, only the high-molecular cleavage product can be seen; the low-molecular product has completely passed through the gel. In addition to the clostridiopeptidase A cleavage products (ME 31 200 and 18 700), row 9 also contains products of unspecific cleavage.
' Abb.6 : Zeitabhängigkeit der Umsetzung der Proteine SA302/1, HS4133/1 und SA506/1 mit Clostridiopeptidase A' Fig.6: Time dependence of the implementation of the proteins SA302 / 1, HS4133 / 1 and SA506 / 1 with clostridiopeptidase A
Die Reaktionsbedingungen sind in Beispiel 3.2 - 3.4 beschrieben. Die Werte für SA506/1 und HS4133/1 wurden mit je 0,01 Einheit und für SA302/1 mit 1,0 Einheit Clostridiopeptidase A erhalten. The reaction conditions are described in Examples 3.2 - 3.4. The values for SA506 / 1 and HS4133 / 1 were obtained with 0.01 unit each and for SA302 / 1 with 1.0 unit of clostridiopeptidase A.

Claims

Patentansprüche Claims
1. Fusionsprotein der allgemeinen Formel I1. Fusion protein of the general formula I
H2N-Z1-X-(Pro-Y-Gly)n-Pro-Z2-COOH (I), in der n ≥ 2 ist,H 2 NZ 1 -X- (Pro-Y-Gly) n -Pro-Z 2 -COOH (I), in which n ≥ 2,
X und Y jede der 20 durch den genetischen Code festgelegten Aminosäuren darstellt,X and Y represent each of the 20 amino acids defined by the genetic code,
Z1 eine bakterielle Aminosäuresequenz undZ 1 is a bacterial amino acid sequence and
Z2 das Zielpeptid aus beliebigen Aminosäuren des genetischen Codes bedeuten.Z 2 is the target peptide from any amino acids of the genetic code.
2. Fusionsprotein nach Anspruch 1, dadurch gekennzeichnet, daß n 2 bis 10 ist und X für Gly steht.2. Fusion protein according to claim 1, characterized in that n is 2 to 10 and X represents Gly.
3. Fusionsprotein nach Anspruch 1 oder 2, dadurch gekennzeichnet, daß Z 1 eine Sequenz von 444 Aminosäuren im Bereich der Aminosäuresequenz 1 - 444 der Präphosphatase aus E.coli K 12 darstel lt. 3. Fusion protein according to claim 1 or 2, characterized in that Z 1 represents a sequence of 444 amino acids in the range of amino acid sequence 1-444 of the prephosphatase from E. coli K 12.
4. Fusionsprotein nach Anspruch 1 - 3, dadurch gekennzeichnet, daß Z2 eine Aminosäuresequenz von humanem ACTH bedeutet.4. Fusion protein according to claims 1-3, characterized in that Z 2 represents an amino acid sequence of human ACTH.
5. Verfahren zur Herstellung von Fusionsproteinen der Formel I, dadurch gekennzeichnet, daß man eine für Proteine der Formel I kodierende Genstruktur in einer bakteriellen Wirtszelle exprimiert und das Fusionsprotein über Trennverfahren erhält.5. A process for the preparation of fusion proteins of the formula I, characterized in that a gene structure coding for proteins of the formula I is expressed in a bacterial host cell and the fusion protein is obtained via separation processes.
6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, daß man als bakterielle Wirtszelle E.coli einsetzt.6. The method according to claim 5, characterized in that E.coli is used as the bacterial host cell.
7. Genstrukturen, kodierend für Fusionsproteine der Formel I.7. Gene structures coding for fusion proteins of the formula I.
8. Plasmide oder vergleichbare Vektoren, die Codons für repetitive Collagenase-Schnittstellen enthalten und damit zur Herstellung von Fusionsproteinen nach Anspruch 1 geeignet sind.8. plasmids or comparable vectors which contain codons for repetitive collagenase interfaces and are therefore suitable for the production of fusion proteins according to claim 1.
9. Plasmid pSA 5069. Plasmid pSA 506
10. Plasmid pHS 413310. Plasmid pHS 4133
11. Verfahren zur Herstellung eines eukaryotischen Proteins, dadurch gekennzeichnet, daß man aus einem Fusionsprotein der Formel I enzymatisch die C-terminal auf die Aminosäuresequenz -X-(Pro-Y-Gly)n-P ro - , in der X und Y jede genetisch kodierbare Aminosäure und n ≥ 2 bedeuten, folgende Proteinsequenz abspaltet. 11. A process for the preparation of a eukaryotic protein, characterized in that enzymatically the C-terminal to the amino acid sequence -X- (Pro-Y-Gly) n -P ro - in which X and Y each from a fusion protein of formula I genetically encodable amino acid and n ≥ 2 mean, the following protein sequence is split off.
12. Verfahren zur Herstellung eines eukaryotischen Proteins nach Anspruch 11, dadurch gekennzeichnet, daß man die Y-Gly-Bindungen in der Aminosäuresequenz -X-(Pro-Y-Gly) -Pro mit einer Collagenase selektiv spaltet und anschließend den N-terminalen Gly-Pro-Rest mit einer Postprol indipeptidylaminopeptidase abspaltet. 12. A method for producing a eukaryotic protein according to claim 11, characterized in that the Y-Gly bonds in the amino acid sequence -X- (Pro-Y-Gly) -Pro are selectively cleaved with a collagenase and then the N-terminal Gly -Pro residue with a postprol indipeptidylaminopeptidase.
EP88907257A 1987-09-18 1988-08-29 Process for producing peptides by specific cleavage of fusion proteins with collagenases obtained by genetic engineering Expired - Lifetime EP0393039B1 (en)

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DE3731874 1987-09-18
DE19873731874 DE3731874A1 (en) 1987-09-18 1987-09-18 METHOD FOR THE PRODUCTION OF PEPTIDES BY SPECIFIC CUTTING OF GENETICALLY OBTAINED FUSION PROTEINS WITH COLLAGENASES

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US5223409A (en) 1988-09-02 1993-06-29 Protein Engineering Corp. Directed evolution of novel binding proteins
US7413537B2 (en) 1989-09-01 2008-08-19 Dyax Corp. Directed evolution of disulfide-bonded micro-proteins
JP4146512B2 (en) * 1991-03-01 2008-09-10 ダイアックス コープ． Small protein
ATE153380T1 (en) * 1993-01-16 1997-06-15 Manfred Schawaller METHOD FOR OBTAINING NATURAL, OLIGOMERIC, GLYCOSYLATED ECTODOMAIN VIRAL MEMBRANE PROTEINS, THEIR USE, IN PARTICULAR AS A VACCINE AGAINST HIV
JP2004531461A (en) * 2000-10-18 2004-10-14 チルドレンズ メディカル センター コーポレーション Osteopontin coated surface and method of use

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EP0095361B1 (en) * 1982-05-25 1989-07-26 Eli Lilly And Company Cloning vectors for expression of exogenous protein
DE3410437A1 (en) * 1984-03-22 1985-09-26 Bayer Ag, 5090 Leverkusen PROCESS FOR PRODUCING PROTEINS
GB8412517D0 (en) * 1984-05-16 1984-06-20 Nagai K Recombinant fusion proteins

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