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EP1214428A1 - Procede de fabrication de serine-proteases actives et de variants inactifs - Google Patents

Procede de fabrication de serine-proteases actives et de variants inactifs

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Publication number
EP1214428A1
EP1214428A1 EP00964151A EP00964151A EP1214428A1 EP 1214428 A1 EP1214428 A1 EP 1214428A1 EP 00964151 A EP00964151 A EP 00964151A EP 00964151 A EP00964151 A EP 00964151A EP 1214428 A1 EP1214428 A1 EP 1214428A1
Authority
EP
European Patent Office
Prior art keywords
serine protease
gzmk
sequence
dipeptide
granzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00964151A
Other languages
German (de)
English (en)
Inventor
Dieter Jenne
Elke Wilharm
Marina A. A. Parry
Wolfram Bode
Robert Huber
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Max Planck Gesellschaft zur Foerderung der Wissenschaften
Original Assignee
Max Planck Gesellschaft zur Foerderung der Wissenschaften
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Filing date
Publication date
Application filed by Max Planck Gesellschaft zur Foerderung der Wissenschaften filed Critical Max Planck Gesellschaft zur Foerderung der Wissenschaften
Publication of EP1214428A1 publication Critical patent/EP1214428A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6467—Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the invention relates to a method for producing biologically active serine proteases and isolated serine protease domains and enzymatically inactive variants of these serine proteases / serine protease domains in prokaryotic hosts.
  • proteases are special proteins with peptidolytic and esterolytic properties and can catalytically change and convert other substances and proteins (substrates) irreversibly. Depending on the functionally relevant molecular residues of the catalytically active center, these proteases are divided into four different main classes: serine-dependent proteases, cysteine proteases, aspartases and metalloproteases. Proteases of the serine type fall into two large families, the family of the actual serine proteases and the subtilisin family.
  • the best-known representatives of the serine proteases include the digestive enzymes of the gastrointestinal tract, the trypsin, chymotrypsin and the pancreatic elastase, the bactericidal and matrix-degrading enzymes of the neutrophilic granulocytes, leukocyte elastase and cathepsin G, the kallikreine of the serum proteases and the salivary glands. and immune system. Serine proteases in secretory granules of mast cells, lymphocytes, phagocytes or natural killer cells and the serine proteases of the complement system play an important role in the immune defense against viruses, parasites, bacteria and tumor cells and in autoimmune processes.
  • Serine proteases specialize in different substrates and can be used for aspartic acid residues (granzyme B, induction of DNA fragmentation in lysed target cells), arginine and lysine residues (trypsin, granzyme A and granzyme K), methionine residues (granzyme M, Met-ase ”) or after hydrophobic amino acids (elastase, proteinase 3, pancreatic elastase, chymotrypsin) hydrolyze a peptide bond.
  • aspartic acid residues granzyme B, induction of DNA fragmentation in lysed target cells
  • arginine and lysine residues trypsin, granzyme A and granzyme K
  • methionine residues granzyme M, Met-ase
  • hydrophobic amino acids elastase, proteinase 3, pancreatic elastase, chymotrypsin
  • lymphocyte-specific serine proteases (called granzymes) are secreted during target cell lysis and are directly and indirectly after uptake into the cytosol of the target cell in the process of target cell destruction activated killer cells involved.
  • Cathepsin G, proteinase-3 and leukocyte elastase are serine proteases from neutrophil granulocytes that break down elastin and other matrix components and are considered as important pathogenicity factors in various chronic inflammatory diseases and autoimmune reactions. Proteinase-3 has also been identified as the disease-specific autoantigen of Wegener's granulomatosis and could be used in the future to treat patients with this condition.
  • Babe (Babe et al. (1998) Biotechnol. Appl. Biochem. 27: 117-124) describes an expression method of a serine protease in a prokaryotic system with secretion into the extracellular medium. However, these authors did not refold the expressed protein and process it with cathepsin C. Furthermore was only a low yield of 200 ⁇ g / l and a low storage stability of the serine protease obtained have been observed.
  • Höpfner Höpfner et al. (1997) EMBO J. 16: 6626-6635) presents the expression of an active serine protease in E. coli, but activation must be carried out in this method by Rüssel's Viper Venom. However, the very sequence-specific enzyme contained in this poison (an endoproteolytically active serine protease) is not generally available. In addition, the signal sequence recognized by the protease is significantly longer and different than the naturally occurring propeptides, which could impair the effectiveness of the renaturation.
  • US Pat. No. 5,679,552 describes the generation of biologically active proteins whose correct N-terminus is achieved by limited proteolysis of an N-terminal helper sequence using cathepsin C.
  • the exact processing of the even-numbered amino acid helper sequence with the help of cathepsin C is achieved in that so-called cathepsin C stop sequences have been artificially inserted into the amino terminus of the protein to be produced.
  • the method is limited to those proteins that have certain sequence properties at the mature N-terminus, i.e. for proteins with lysine or arginine in the first position, or for proteins with proline in the second or third position within the sequence of the protein to be produced.
  • Serine proteases with the N-terminal consensus sequence Ile- (Ile / Val) -Gly-Gly cannot be produced using the method described in US5679552.
  • the highly conserved N-terminal sequence of functionally active serine proteases does not correspond to any of the previously known cathepsin C stop sequences.
  • EP 0397420 protects the enzymatic conversion of recombinant proteins with helper sequences using cathepsin C using a novel N-terminal stop sequence (Met-Tyr and Met-Arg).
  • the N-terminal end (Met-Tyr or Met-Arg) of the recombinant protein to be produced which is described for the first time in this patent, cannot be cleaved off by exopeptidases such as cathepsin C.
  • the stop sequences shown in patent EP 0397420 are Also unsuitable for processing and displaying a functional N-terminus in catalytically active serine proteases.
  • US Pat. No. 5,013,662 describes the production of N-terminal methionine-free proteins in E. coli.
  • the N-terminal methionine caused by the start codon is cleaved by methionine aminopeptidase in vitro or in vivo.
  • the N-termini of serine proteases (Ile- (He / Val) -Gly-Gly) cannot be produced by this method either, since the methionine aminopeptidase methionine only cleaves off small amino acids (Gly, Val, Ser, Ala) but before large, aliphatic amino acids such as the absolutely necessary isoleucine.
  • the object of the present invention is therefore to provide a method which can be used in the production of biologically active serine proteases or serine protease domains and catalytically inactive but correctly folded variants in prokaryotic hosts.
  • the method was not developed for the direct synthesis of natural serine proteases with a complex domain composition (coagulation factors, complement proteases), but specifically for simple serine proteases that only consist of a single domain, the catalytic table, and therefore only a specific one Exercise peptidolytic or esterolytic function with natural or artificial specificity (designer activities)
  • These simple serine proteases are widespread in nature and have extremely different tasks in the field of cellular and humoral immune defense (mast cell, granulocyte and lymphocyte proteases, complement factor D), gastroenteral digestion (trypsins, chymotrypsin and elastases), exocrine and endocrine organs (kallikreine), for the normal physiology of the skin and nervous system (neuropsin,
  • the method is suitable for producing such serine proteases and derived therefrom Serine proteases with artificial substrate specificity on an industrial scale.
  • the products can thus be inexpensively provided in unlimited quantities as research reagents, therapeutic agents and for inhibitor development and testing of these inhibitors in vivo.
  • the present invention thus relates to a method for producing biologically active serine proteases, isolated serine protease domains and their amino acid variants in a prokaryotic host, which is characterized by the following steps
  • serine proteases are understood to mean all those proteins which have a structural similarity to trypsin and chymotrypsin. These include, for example, the serine proteases of the coagulation and complement system, the immune defense cells, the gastrointestinal tract and the exocrine glands.
  • the term serine protease domain refers to independently foldable parts of complex proteins with a structural, three-dimensional similarity to serine proteases. These serine protease domains predominantly have peptidolytic and esterolytic properties, but sometimes also other functions.
  • the serine proteases of the coagulation and complement system consist of different covalently linked protein domains and a carboxy-terminally located serine protease domain with catalytic properties. Correctly processed N-terminus, which generally begins with an isoleucine or valine, is essential for the activation and thermodynamically stable folding of serine proteases.
  • Prokaryotic hosts which can be used in the sense of the invention are known to the person skilled in the art and include, inter alia, organisms such as Escherichia, Bacillus, Erwinia and Serratia species, in particular E. coli, Bacillus subtilis, Erwinia chrysanthemi, Erwinia carotovora or Serratia marcescens.
  • organisms such as Escherichia, Bacillus, Erwinia and Serratia species, in particular E. coli, Bacillus subtilis, Erwinia chrysanthemi, Erwinia carotovora or Serratia marcescens.
  • the following can preferably be used: E. coli, Bacillus subtilis.
  • helper sequence at the amino terminus of serine proteases or serine protease domains is carried out with the help of cloning techniques and genetic engineering manipulations in prokaryotic cells, DNA molecules or parts of these molecules being introduced into plasmids and optionally by targeted mutagenesis and recombination of DNA segments can be adapted to the required sequence.
  • PCR polymerase chain reaction
  • vector constructs can be produced which lead to the expression of serine proteases with N-terminal helper sequences.
  • adapters or linkers can be added to the fragments to be cloned.
  • Appropriate restriction interfaces can also be inserted or redundant, e.g. will remove non-coding DNA or unwanted restriction sites. If insertions, deletions or substitutions are desired, the techniques of in vitro mutagenesis, repair using modified primers, PCR, restriction digestion and ligation are used.
  • the degeneration of the genetic code offers the expert, inter alia, the possibility of adapting the nucleotide sequence of the DNA sequence to the codon preference of the respective prokaryotic host. Restriction digestion, sequencing and other biochemical-molecular biological tests are required as analytical methods for assessing the work results.
  • the desired sequence to be expressed can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components.
  • synthetic DNA sequences are generated with codons, which are preferred by prokaryotes. These codons, which are preferred by prokaryotes, can be found in published tables (http://pegasus.uthct.edu) and are most frequently found in highly expressed endogenous proteins.
  • codons which are preferred by prokaryotes
  • different DNA subfragments can be manipulated and combined individually to obtain a DNA sequence that is equipped with a correct reading frame and is translated in the correct direction.
  • adapters or linkers can be used for the simplified linking of DNA fragments.
  • molecular biological DNA vectors with special control areas which control the transcription of the expression cassette in prokaryotic cells.
  • control areas usually include the promoter and special regulatory elements.
  • These regulatory elements such as the tac-lac, 11 or trp promoter, are familiar to the person skilled in the art:
  • Appropriate prokaryotic expression vectors can be obtained from various companies: pET24c and other pET vectors from Novagen, lambda gtl l and pGBT9 from Clontech, pGX from Qiagen.
  • “Expression of the serine protease (s)” in the sense of the invention is understood by the person skilled in the art to mean the expression of a heterologous fusion protein in the prokaryotic host.
  • the process for the preparation of serine proteases and / or their fragments includes the expression of a proform in the cytosol of the prokaryotic host, optionally and preferably as inclusion bodies ("IB," inclusion bodies ").
  • IB inclusion bodies
  • the inclusion body formation depends primarily on the expression rate, whereby there is no clear correlation with the size, hydrophobicity and other properties of the protein to be expressed (Lilie, H. et al.
  • the invention therefore also includes a method in which the expression is carried out as soluble recombinant protein / peptides.
  • “Renaturation of the expressed fusion proteins” in the sense of the invention is understood to mean the solubilization of protein aggregates and unfolding into naturally identical three-dimensional structures which are stable in physiological buffer solutions.
  • the method according to the invention further comprises activating the serine proteases to be produced or their serine protease domains by cleaving off a suitable helper sequence by means of an exopeptidase.
  • exopeptidases are particularly preferably as detailed below. Examples include: Cathepsin C, Cathepsin W, Cathepsin B or Diaminopeptidase IV, Cathepsin C-like functional homologues in other species such as Dictyolstelium diseoideum and C. elegans.
  • exopeptidase substrates to be processed they do not carry the known exopeptidase stop sequences either in the region of the helper sequence or in the amino-terminal region of the desired product.
  • a sequential conversion by a convenient one A combination of different exopeptidases could be carried out, in which, for example, methionine was split off via a specific methionine aminopeptidase in the first step and dipeptide (s) were removed in further steps.
  • dipeptides or a combination of several different dipeptides from a pool of suitable dipeptides without natural stop sequences are additionally connected upstream of the N-terminus of the serine protease to be expressed.
  • These peptide helper sequences encompass any amino acid combination, but proline, lysine or arginine cannot be in the first place and proline cannot be in the second place of this dipeptide.
  • a dipeptide of the form Met-X for example Met-Glu
  • step (a) the sequence of the desired serine protease product, which in step (g) is carried out by self-limited conversion using cathepsin C is split off.
  • the N-terminally added dipeptide begins with a methionine. Because the translation in prokaryotes begins with a formyl-methionine residue, which in some cases is associated with the endogenous E. coli formylase in the cytosol of the bacteria the £.
  • the methionine-containing dipeptides (Met-Y) added in this preferred embodiment are on the one hand resistant to post-translational processing in E. coli, but on the other hand a good substrate for dipeptidylaminopeptidases previously only detected in eukaryotes
  • sequences of the form (Met) n-Glu such as Met-Met-Gly (where n is a natural number up to 40 mean)
  • Met-Met-Gly where n is a natural number up to 40 mean
  • methionines can be used during or after expression, i.e. hm vitro, to be cleaved by methionine aminopeptidase (s)
  • care must be taken to ensure that after cleavage of the methionine (s) a helper sequence occurs which has an even number of amino acids without stop amino acids.
  • the dipeptide helper sequence Met-Glu is particularly suitable for the embodiment proposed above, it being possible for this dipeptide helper sequence to be preceded by one or more methionines.
  • Proline should be avoided at the second position of the dipeptide, as it prevents the dipeptide from being split off by individual exopeptidases, such as cathepsin C. It should be mentioned that tyrosine or arginine should also be avoided at the second position after methionine if cathepsin C is to be used as the exopeptidase in the process according to the invention.
  • the dipeptide Met-Glu is used as the helper sequence and the conversion enzyme cathepsin C.
  • the present invention thus relates to a method in which, in the preferred embodiments, the exopeptidase is a monoaminopeptidase and / or a diaminopeptidase.
  • monoaminopeptidase is understood to mean the above-mentioned methionine aminopeptidase from E. coli (Ben-Bassat et al. (1987) J. Bacteriol. 169: 751-757).
  • the diaminoexopeptidase is cathepsin C or a cathepsin C homolog (RCP, described in US Pat. No. 5,637,462).
  • the invention relates to a deterioration as described above, wherein the biologically active serine proteases and serine protease domains to be produced do not inactivate the exopeptidase to be used and, in a further embodiment, the exopeptidase does not irreversibly change the protein to be produced. Inactivation of the exopeptidase (s) by active serine proteases (e.g. by proteolytic cleavage) should be avoided.
  • an irreversible change in the serine protease (s) and / or its fragments to be produced should be understood to mean cleavages, changes in conformation and / or inhibitions.
  • aggregate formation between the proteins / fragments to be produced and the exopeptidase used should be avoided.
  • the invention comprises a method, wherein the fragment of a serine protease to be produced is the catalytic domain of a serine protease.
  • the invention further comprises a method in which the biologically active serine protease to be produced is seen from one or more non-covalently linked catalyti
  • the invention also encompasses those serine proteases and their fragments which are due to
  • Serine protease variants occur naturally and other non-catalytic ones
  • the invention also includes mutine-derived serine proteases without catalytic activity.
  • the invention comprises a method in which the
  • Serine protease leukocyte elastase proteinase-3, complement factor D, azurozidine,
  • the invention relates to a method for producing a granzyme, the granzyme being A, B, K, H, M or L.
  • Granzyme L comprises the nucleotide sequence shown here in SEQ ID No. 1.
  • the vector pET24c (Novagen) was cut for the cloning of human granzyme K with the restriction endonucleases Ndel and EcoRI.
  • the sequence coding for human granzyme K was amplified by means of two-stage PCR from human bone marrow cDNA.
  • the human granzyme K cDNA was amplified between the middle of the first and the end of the fifth exon of human bone marrow cDNA using a pair of correctly hybridizing oligonucleotides (so-called outer oligos, see P1 and P2 in the sequence listing)
  • PCR Conditions template DNA: 1 ⁇ l of an mRNA transcribed into cDNA with reverse transcriptase (2 ⁇ g), nucleotides: 0.2 mM each; oligos: 1 ⁇ M each; enzyme 0.5 ⁇ l / 50 ⁇ l mixture [2.5 units / ⁇ l] native yw- Polymerase (Stratagene); buffer: Stratagene; program: non-cyclic denaturation: 5 minutes at 95 ° C, cyclical steps: 1 minute at 95 ° C, 1 minute at 56 ° C, 1 minute at 72 ° C, 35 cycles, non-cyclic elongation: 5 minutes at 72 ° C).
  • PCR product obtained served as a template sequence in the second round of PCR, in which the insert for the cloning into the pET24c vector was amplified by means of a second pair of oligonucleotides (inner oligos, see P3 and P4 in the sequence listing)
  • PCR conditions template DNA: 5 ng; oligos: 1 ⁇ M each; nucleotides: 0.2 mM each; enzyme: 0.5 ⁇ l / 50 ⁇ l batch [2.5 units / ⁇ l] native w-polymerase (Stratagene); buffer: Stratagene; program: non-cyclic denaturation: 5 minutes at 95 ° C, cyclical steps: 1 minute at 95 ° C, 1 minute at 58 ° C, 1 minute at 72 ° C, 24 cycles, non-cyclic elongation: 5 minutes at 72 ° C).
  • the Ndel and EcoRI interfaces were introduced into the oligos.
  • the amplificate obtained in this way was purified from the gel (Qiaquick protocol from Qiagen) with the restriction endonucleases Ndel and EcoRI and cut into the vector (ligation conditions: enzyme: 1.5 ⁇ l / 20 ⁇ l mixture [1 unit / ⁇ l] T4 ligase (Boehringer Mannheim); vector: 50 ng / 20 ⁇ l mixture; insert: 50 ng / 20 ⁇ l mixture; buffer: Boehringer Mannheim; incubation: 16 hours at 15 ° C), so that 8 bases in 3 'direction from the ribosome binding site translate the Transcript begins with the methionine of the N ⁇ el palindrome.
  • the ⁇ -terminal sequence for both granzymes is composed of the pro (di) peptide Met-Glu and the subsequent conserved sequence of the mature granzymes Ile-Ile-Gly-Gly.
  • the translation stops with the natural stop codon.
  • Granzyme K of the mouse was amplified using the identical oligonucleotide in the ⁇ -terminal region (P5 and P3) and P6 as Reverse primer.
  • the granzyme K fragment of the mouse for the expression cassette was amplified in 35 cycles using 5 ng DNA, purified from the gel (Qiaquick protocol from Qiagen) with the restriction endonuclease Ndel and the 3'- End kinased.
  • PCR 0.2 mM of the four nucleotides, 1 ⁇ M of each oligo and 0.5 ⁇ l / 50 ⁇ l batch [2.5 units / ⁇ l] native / w polymerase (Stratagene) in the buffer system from Stratagene and the following thermocycler program were used: non-cyclic Denaturation: 5 minutes at 95 ° C, cyclical steps: 1 minute at 95 ° C, 1 minute 51 ° C, 1 minute 72 ° C, 35 cycles, non-cyclic elongation: 5 minutes at 72 ° C, for cloning in pET24c became the vector by digestion with Hind ⁇ .
  • the insert was ligated into the vector thus prepared as described above.
  • the resulting clones were selected for kanamycin [30 ⁇ g / ml] and verified by restriction analysis (double digestion) using Ndel and Xh ⁇ l (New England Biolabs) and sequencing.
  • the vector pET24c-His-Strep-tag (modified Novagen vector) was cut for the cloning of human granzyme M with the restriction endonucleases Ndel and Pstl.
  • the sequence coding for human granzyme M was amplified by means of two-stage PCR from human bone marrow cDNA.
  • the human granzyme M cDNA was amplified between the middle of the first and the end of the fifth exon of human bone marrow cDNA using a pair of correctly hybridizing oligonucleotides (so-called outer oligos, see P7 and P8 in the sequence listing)
  • PCR Conditions Template DNA: 1 ⁇ l of an mRNA transcribed into cDNA with reverse transcriptase (2 ⁇ g); nucleotides: 0.2 mM each; oligos: 1 ⁇ M each; enzyme 0.5 ⁇ l / 50 ⁇ l mixture [2.5 units / ⁇ l] native w -Polymerase (Stratagene); buffer: Stratagene; program: non-cyclic denaturation: 5 minutes 95 ° C, cyclic: 1 minute 95 ° C, 1 minute 56 ° C, 1 minute 72 ° C, 24 cycles, non-cyclic elongation : 5 minutes 72 ° C).
  • the PCR product obtained served as template DNA in the second round of PCR, in which, using a second pair of oligonucleotides (inner oligos, see P9 and P10 in the sequence listing), the insert for cloning into the pET24c-His-Strep-t ⁇ g vector was amplified (PCR conditions: template DNA: 5 ng; oligos: 1 ⁇ M each; nucleotides: 0.2 mM each; enzyme: 0.5 ⁇ l / 50 ⁇ l mixture [2.5 units / ⁇ l] native PW polymerase (Stratagene); buffer : Stratagene; program: non-cyclic denaturation: 5 minutes 95 ° C, cyclic: 1 minute 95 ° C, 1 minute 58 ° C, 1 minute 72 ° C, 24 cycles, non- cyclic elongation: 5 minutes 72 ° C).
  • PCR conditions template DNA: 5 ng; oligos: 1 ⁇ M each; nucleotides: 0.2
  • the Ndel and Nsil interfaces were introduced in the oligos.
  • the sequence coding for the first ten amino acids was additionally optimized with regard to the codon frequency in E. coli (Fig. 3).
  • the influence of this codon optimization on the expression strength is illustrated in Fig. 2b.
  • the amplificate obtained in this way was purified from the gel (Qiaquick protocol from Qiagen) and ligated into the vector (ligation conditions: see example la).
  • the pro (di) peptide Met-Glu is also used as the N-terminal helper sequence for mouse granzyme K and the cDNA reading frame of the mature murine granzyme K is added to it.
  • translation with the natural stop codon stopped.
  • the resulting clones were selected for kanamycin [30 ⁇ g / ml] and confirmed by restriction analysis (double digestion) with Ndel and EcoRI (New England Biolabs) and DNA sequencing.
  • the plasmids constructed according to examples la and lb were transformed into the expression strain E. coli B834 DE3 (Novagen) and the expression was first tested on a small scale. 10 ml cultures were grown with LB-kanamycin (for concentration see Example 1) to an OD600 of 0.5, the expression was induced with 1 mM IPTG and incubated for 3 hours at 37 ° C. to an OD600 of 1.5. IPTG activates the IacUV-Promoto ⁇ , which controls the chromosomally encoded T7 polymerase in the B834 DE3 strain, which in turn takes on the transcription of the cloned granzyme gene under T7 / c promoter control.
  • the bacteria were harvested by centrifugation and the pellet washed once with PBS pH 7.4 before digestion in lysis buffer at room temperature.
  • the bacterial membranes were broken either by two French press cycles (1000-1200 psi) or three sonification cycles (15 minutes each 320 W), the lysate was mixed with a third of the volume of wash buffer I and incubated for 1 hour at room temperature in an overhead shaker , The suspension was centrifuged at 17200 g at 4 ° C. for 20 minutes, the pellet was resuspended in washing buffer I, incubated for 1 hour at room temperature in a shaker and centrifuged again. This process was repeated twice with wash buffer I and three times with wash buffer II. After the last centrifugation, a small aliquot of the IB preparation was analyzed for purity using SDS-PAGE, the rest was solubilized. The following buffers were used for this:
  • the renaturation was carried out in three pulses with time intervals of 8 hours each.
  • the renaturation batches of human granzyme K were incubated at room temperature, those of mouse granzyme K and human granzyme M at 4 ° C.
  • the protein solution (-10 mg / ml) from Example 2 was in each case diluted 1: 100 (vol / vol) in the renaturation buffer with stirring and incubated until the next pulse without agitation.
  • the refolding batch was incubated for a further two days at room temperature or 4 ° C. without agitation.
  • the reaction volume was filtered (via cellulose acetate), concentrated to approx.
  • Cathepsin C was activated in the presence of a thiol component and halide ions by reduction, for example by 10 mM mercaptoethanolamine HCl. Since it is through the However, if a reducing agent is present in the conversion mixture, the disulfide bridges of the folded granzyme can be reduced, the activation and conversion conditions were first optimized in terms of the thiol concentration, the duration of activation and the subsequent dialysis, and the pH. The optimal parameters used were 2 mM mercaptoethanolamine for activation at pH 5.0 over 20 minutes and then dialyzed against PBS, 75 mM NaAcetat at pH 5.5 for 20 minutes.
  • the FPLC peak fraction was dialyzed against PBS pH 6.0 and concentrated to approx. 1 ml ( ⁇ 20 mg protein / ml). 3 units of cathepsin C per milliliter [stock: 5U / ml in H2O] were initially for 20 minutes at 37 ° C. in 5 mM 2-mercaptoethanolamine,
  • PBS pH 5.0 activated and then dialyzed for 20 minutes at room temperature against PBS, 75 mM Na acetate, pH 5.5 to remove 2-mercaptoethanolamine.
  • the active cathepsin C was mixed 1: 1 (vol / vol) with the zymogen and incubated for 6 hours at room temperature, any precipitations that occurred were separated by centrifugation and the filtered sample was subjected again to cation exchange chromatography (see Example 3) to separate cathepsin C.
  • the thiobenzyl ester substrates and Ellman's reagent were diluted to final concentrations of 0.3 mM in test buffer (150 mM Tris, 50 mM NaCl, 0.01% Triton X-100, pH 7.6).
  • the various granzyme preparations were also diluted to 3-15 nM in test buffer.
  • the color change associated with the conversion of the substrates was measured at 405 nm and room temperature over 5 minutes in an ELISA reader. To calculate the turnover rate, the difference between the absorptions at the beginning and after 5 minutes was formed and related to the time.
  • Fig. 1 Description of the primers used
  • Fig. 2a Analysis of the expression, purification and renaturation of human
  • Fig. 2b Analysis of the expression of human granzyme M (hGzmM) using different expression constructs. 12% SDS-PAGE with subsequent Coomassie staining of hGzmM with natural C-terminus (name hGzmM / WT) and (His) 8-Strep-t g-C-terminus (name
  • the digestion procedures are: 1: non-induced culture, digestion in 2.5% SDS, 5% b-mercaptoethanol; 2: induced culture, digestion as in 1; 3: induced culture, digestion in 5% SDS, 200 mM DTT, 5% b-mercaptoethanol; 4: induced culture with idealized codons, digestion as in 1.
  • Fig. 3 N-terminal sequence comparison between human granzyme K (hGzmK) and human granzyme M (hGzmM) at the amino acid level (A) and nucleotide level (B).
  • the codon frequencies in E. coli (Ausubel et al., 1999) are given in%, rare codons are underlined.
  • the one for N-terminal optimization the oligo designed for expression constructs is shown in (C), the changed positions are underlined.
  • Fig. 4a Substrate specificity of human granzyme K (hGzmK). 0.1 mM each of the thiobenzyl ester substrates and 3 nM each of the proform (black), the converted form (hatched) and the converted S195A mutant (gray) were used. The substrate conversion was measured at 405 nm and room temperature over 5 minutes.
  • Fig. 4b Substrate specificity of mouse granzyme K (mgzmK). 0.1 mM each of the thiobenzyl ester substrates and 5 nM each of the proform (black) and the converted form (hatched) were used. The substrate conversion was measured at 405 nm and room temperature over 5 minutes.
  • Fig. 4c Substrate specificity of human granzyme M (hGzmM). In each case 0.1 mM of the thiobenzyl ester substrates and 15 nM of the Proform
  • Fig. 5 Granzyme L cDNA sequence
  • Fig. 6 Amino acid sequence of granzyme L. Generation of catalytically active granzyme K from Escherichia coli inclusion bodies and identification of efficient granzyme K inhibitors in human plasma
  • Granzymes are granule-stored lymphocyte serine proteases that are involved in cytotoxic defense reactions of T and natural killer cells after target cell recognition.
  • a fifth human granzyme (granzyme 3, lymphocyte tryptase-2), renamed granzyme K (gene name GZMK), has recently been cloned from lymphocyte tissue.
  • GZMK granzyme 3
  • the natural proform of granzyme K with the amino-terminal propeptide Met-Glu was expressed in the form of inclusion bodies and, after refolding of precursor molecules, converted to its active enzyme by cathepsin C.
  • Recombinant granzyme K cleaves synthetic thiobenzyl ester substrates according to Lys and Arg with k cat / K m values of 3.7 x 10 and 4.4 x 10 4 M "1 s * 1. It has been shown that the activity of granzyme K by the synthetic compounds Phe-Pro-Arg-chloromethyl ketone, phenylmethylsulfonyl fluoride, PefablocSC and benzamidine, which are inhibited by the Kunitz-type inhibitor aprotinin and by human plasma.
  • the subunit and the second carboxy-terminal Kunitz type domain of bikunin have been identified as true physiological inhibitors with Kj values of 64, 50 and 22 nM, respectively.
  • Inter- ⁇ -trypsin inhibitor and free bikunin have the potential to be extracellular Neutralize the activity of granzyme K after T cell degranulation, and could thus contain non-specific damage to neighboring cells at locations of inflammatory reactions.
  • Perforin and Granzyme are essential components of cytosolic granules and fulfill important tasks for the secretion-dependent cytotoxicity mediated by T cells and natural killer cells against virus-infected host cells, tumor cells, and antigenetically modified non-transformed host cells. After specific recognition of the target cell, granules are exocytosed in the direction of the target cell membrane, and perforin and granzymes are released in concerted action (1-4).
  • the two quantitatively most important granzymes of cytolytic T cells are granzymes A and B, which have been shown to contribute to the induction of apoptosis and DNA fragmentation in target cells.
  • All granzymes are synthesized as pre-pro-enzymes in the rough endoplasmic reticulum and into active enzymes in a two-step process by cleavage of the signal peptide and subsequent removal of the propeptide by means of a quite similar, presumably identical dipeptidylaminopeptidase from cytosolic granules, called cathepsin C, converted (15, 16).
  • GzmK granzyme 2, lymphocyte tryptase-2
  • granzyme 2 lymphocyte tryptase-2
  • RNK-16 rat NK tumor line
  • the human GzmK has recently been cloned and has been shown to have hydrolytic activities for the thioester substrates Z-Lys-SBzl and Z-Arg-SBzl (21) and for a highly basic peptide which is the SV40 nuclear localization signal (24), has. High mRNA levels for GzmK are detected in NK cells and activated T cells, but are absent in normal tissues that do not contain a high number of these cells.
  • Recombinant granzymes have been produced by secretion and transport of precursor forms that carry signal sequences in various eukaryotic and prokaryotic host systems, including yeast (25), baculovirus-infected insect cell lines (26), mammalian cell lines (16, 27), Escherichia coli (28), and Bacillus subtilis (24).
  • yeast 25
  • baculovirus-infected insect cell lines 26
  • mammalian cell lines (16, 27)
  • Escherichia coli 28
  • Bacillus subtilis Bacillus subtilis
  • Amino-terminally extended and catalytically inactive precursors of GzmK are folded back into soluble proteins and then subjected to limited proteolysis by reaction with a dipeptidyl aminopeptidase, cathepsin C, which appeared suitable for selectively removing the excess amino acids at the amino terminus, which are in catatalytically active GzmK are not available.
  • I ⁇ l inter- ⁇ -trypsin inhibitor
  • the cDNA coding for the human GzmK was determined by means of PCR of bone marrow cDNA using the outer oligonucleotides DJ 209 5'-TTC CTA ATA GTT GGG GCT TAT-3 (coding strand) and DJ 210 5'-CAA CTC TAA CCT GCG AGC ATA-3 '(counter strand) amplified.
  • Inner oligonucleotides DJ 255 (5 " -GGC TTA CCA TAT GGT TAT TGG AGG GAA AGA A-3), DJ 601 (5'-TGT GTT TCC ATA TGG AAA TTA-3 1 ), DJ 373 (5'-GGC TTA CCA TAT GGG GGA AAT TAT TGG AGG G-3 ') (coding strand) introduced an Nde I restriction site and the rear oligonucleotide DJ 535 (5'-AAT AGA ATT CTT TGT AAC TTA ATT-3', opposite strand) introduced an Eco RI restriction site (Fig -f)
  • the product of the amplification with DJ 373 / DJ 535 includes the N-terminal prosequence Met-Gly-Glu, the amplificate with DJ 601 / DJ 535 starts with the natural propeptide sequence Met-Glu from human GzmK (Fig.
  • the bacteria were lysed in 50 mM T ⁇ s-HCl, 2 M MgCl 2 with 10 ⁇ g / ml DNase I and 0.25 mg / ml lysozyme, pH 7.2 by French Press or ultrasound treatment.
  • IB were separated by centrifugation and twice with 50 mM Tns-HCl, 60 M EDTA, 1.5 M NaCl, 6% Triton X-100, pH 7.2, followed by two washes with 50 M Tns-HCl, 60 mM EDTA, pH 7.2
  • the purified EB were in 6 M guanidinium hydrochloride, 100 mM Tns-HCl, 20 mM EDTA, 15 mM GSH, 150 mM GSSG, pH 8.0 solubilized overnight at room temperature (RT) in an overhead shaker and then at 4 ° C.
  • the dialysate was centrifuged at 30000xg, filtered and, for further purification and concentration, via rapid protein liquid chromatography (FPLC). applied to a Mono S Sepharose column (Amersham Pharmacia Biotech) GzmK was eluted from the column at RT in a linear salt gradient of 20 column volumes from 0.15 to 2 M NaCl in the same buffer (PBS) and the protein concentration of the GzmK fraction was determined using the Bradford (Coomassie binding) detection.
  • FPLC rapid protein liquid chromatography
  • the FPLC fractions of various refolded GzmK proforms were buffered by dialysis against PBS, pH 6.5 and concentrated to approx. 10 mg / ml.
  • Bovine Cathepsin C (Sigma) was activated for 30 minutes at 37 ° C in PBS, 10 mM 2-mercaptoethanolamine, 75 mM Na acetate, pH 5.0 and 30 minutes against PBS, 75 mM Na- before incubation with GzmK proforas. Acetate, pH 5.5 dialyzed. Five units of cathepsin C in dialysis buffer and 10 mg GzmK-Proform were mixed in equal volumes and incubated at RT.
  • the increase in enzymatic GzmK activity was measured as described below using the synthetic substrate N ⁇ -benzyloxycarbonyl-L-lysine-thiobenzyl ester (Z-Lys-SBzl) and the incubation was stopped with constant activity.
  • the active GzmK was separated from Cathepsin C using the cation chromatography described above.
  • the concentration of the active GzmK was then determined by titration of the active centers with 4-nitrophenyl-4'-guanidinobenzoate (4-NPGB) (Sigma).
  • the concentration of Bikunin, the second Kunitz domain of Bikunin and I ⁇ l were determined by titration with trypsin, which was also titrated with 4-NPGB.
  • Activity tests were carried out at RT in a 96 well with a reaction volume of 150 ⁇ l / well in 50 mM Tris-HCl, 0.15 M NaCl, 0.01% Triton X-100, pH 7.6 with 0.3 mM 5.
  • 5'-dithiobis (2-nitrobenzoic acid) (Ellman's reagent) (Sigma).
  • the substrate concentrations were 0.3 mM in the activity tests and between 0.05 and 1.5 mM in kinetic studies.
  • Z-Lys-SBzl (Sigma) was dissolved in water, Z-Arg-SBzl, Boc-Ala-Ala-Met-SBzl and Boc-Ala-Ala-Asp-SBzl (Enzyme Systems Products, Livermore, CA, USA) Me 2 SO.
  • GzmK was used after titration of the active centers in concentrations of 3 nM in activity tests and 0.5-7.5 nM in kinetic tests.
  • the hydrolysis rate of the substrates was determined as a measure of the activity, and the increase in absorption at 405 nm over time was measured in the Dynatech MR4000 ELISA reader.
  • Equilibrium dissociation constants Kj in the case of reversible GzmK inhibitor interactions and constants of the association rate (k. ⁇ ss ) of GzmK with antithrombin III (ATIII) were determined as described elsewhere (32).
  • aprotinin bovine pancreas trypsin inhibitor
  • Bayer Bayer (Leverkusen) GzmK was pre-incubated for 15 minutes at RT with different concentrations of each inhibitor before the reaction was started with the addition of the Z-Lys-SBzl substrate.
  • the decrease in the hydrolysis rate was measured and Kinetic constants calculated by non-linear regression In plasma tests, filtered (0 22 ⁇ m) EDTA blood plasma from a young and healthy donor was treated with 3 nM titrated GzmK and pre-incubated for 15 minutes at RT before substrate addition.
  • ⁇ 2 - Macroglobulin was chemically inactivated by 4 - Incubation of the freshly isolated blood plasma at 23 ° C.
  • the I ⁇ l concentration in the plasma samples used was determined by radial immunodiffusion. For this purpose, holes of 2 mm in diameter were punched in horizontal agarose gels, which consisted of 3% PEG 4000, 1% SeaKe agarose (FMC Bioproducts, Rockland, ME, USA), 0 02% sodium azide, 16 8 mg / 1 rabbit anti-human I ⁇ l (DAKO, Glostrup, Denmark) in 25 mM barbital buffer (3 7 M diethyl barbiturate, 21 3 M 5.5 diethyl barbiturate sodium salt, 1 3 mM calcium lactate, 0 7% sodium azide , pH 8 6) were poured into the punches in T ⁇ or duplicates 5 ⁇ l of the different plasma samples or different dilutions of the titrated I ⁇ l as standard.
  • the gels were incubated at 4 ° C. in a moist chamber until the formation of rings was completed , then dried and stained in 0.1% amido black, 45% methanol, 2% acetic acid for 3-5 minutes. After decolorization in 90% methanol, 2% acetic acid, a standard curve was created from the areas of the titrated I ⁇ l samples from which the I ⁇ l concentrations of the plasma samples were determined
  • coli either started with a methionine followed by the modified N-terminus Val-Ile-Gly-Gly or consisted of Met-Gly-Glu or Met-Glu followed by the natural amino acid sequence of mature GzmK, Ile-Ile-Gly-Gly (Fig.?).
  • E. coli strain B834 DE3
  • high expression rates were achieved with yields of 50-75 mg IB per liter culture or a 50% share of the total bacterial protein. Due to the high yields and insolubility of the recombinant proteins, the IB could be efficiently removed from the £. co / Z proteins are separated and subjected to refolding without further purification steps.
  • S shows total bacterial extracts before and after induction of protein expression as well as an IB preparation. Proteins that remained soluble after replacing the refolding buffer with PBS were analyzed using SDS-PAGE and showed the expected size of GzmK. The degree of methionine cleavage in E. coli by endogenous MAP was examined by N-terminal sequencing of the renatured, FPLC-purified proforms and estimated from the ratio of the two phenylthiohydantoin derivatives released at each step of cyclic Edman degradation. The methionine of the pre-sequence Met-Gly-Glu was only partially removed, so that only two thirds of this Proform started with the desired dipeptide Gly-Glu.
  • the GzmK pro forms obtained in this way were then subjected to the exopeptidolytic activity of bovine cathepsin C.
  • Amino-terminal protein sequencing confirmed the successful conversion of both proforms, Gly-Glu-GzmK and Met-Glu-GzmK. Amino acid residues of shorter sequences were not detected in the Edman degradation.
  • proform construct with the Met-Glu dipeptide sequence at the amino terminus of the mature GzmK represents the most favorable design of a proform that can be processed in vitro using the dipeptidylaminopeptidase cathepsin C into an authentic enzyme.
  • Glutamate in the second position prevents the cleavage of amino terminal methio residues in E. coli and, together with methionine, is a suitable in wtro substrate for bovine cathepsin C.
  • Met-Glu proforms for GzmK 20% of the IB material was folded back and subjected to conversion Half of it was finally obtained in highly concentrated and homogeneous form after ion exchange chromatography
  • Effective plasma inhibitors exist for most granule-associated serine proteases such as leukocyte elastase, cathepsin G and proteinase 3 (35).
  • GzmK-specific inhibitors a constant amount of recombinant GzmK was incubated with increasing concentrations of whole plasma and the Z-Lys SBzl esterase activity measured GzmK was inhibited by plasma proteins even in low concentrations in a dose-dependent manner (Fig i?
  • ATIII showed no inhibitory potential in concentrations corresponding to the ATIII concentrations in the full plasma tests ( Fig 1 3 bars) Since inhibition of GzmK by ⁇ 2 -macroglobulin (data not shown) and ATIII in the absence of heparin does not appear to be responsible for the main inhibitory capacity of human plasma, we preferred I ⁇ l, the predominant form of bikunin in human plasma, as another possible inhibitor for human GzmK into consideration.
  • Bikunin D2 The carboxy-terminal Kunitz-type domain of Bikunin (Bikunin D2) is known to inhibit trypsin-like enzymes with Lys and Arg specificity at the S1 secondary position, but an endogenously synthesized target enzyme with high affinity for this domain has not yet been identified .
  • Bikunin D2 which was produced in recombinant form in Pichia pastoris, and determined its aquilibrium dissociation constant (K;). Domain 2 efficiently blocked GzmK activity with a K; of 22 nM.
  • a properly designed amino terminus starting with I (IV) GG is critical for a granzyme to take its active conformation.
  • the amino-terminal methionine is only removed by the endogenous MAP if amino acid residues are smaller Great ones like glycine, alanine, senn, threonine, valine, or proline follow at the second position (34).
  • Our initial attempts to solve this problem for GzmK were unsuccessful. Valine naturally occurs at the amino terminus of some serine proteases and was therefore considered a viable replacement for the first Ile residue, but when we replaced He to favor the removal of amino-terminal Met residues, over 90% of the recombinant molecules still carried the methionine residue as we did by amino-terminal sequencing.
  • the procedure described here is the first one that is about aggregate £. coli inclusion bodies enable the preparation of active human GzmK and its natural precursor by refolding and exopeptidolytic processing.
  • the enzyme shows the same biochemical properties and specificities as biosynthetically folded GzmK, which originates from lymphokine-activated killer cells or from supernatants from GzmK-secreting B. subtilis (21, 24).
  • Our process delivers pure and active enzyme in the milligram range and thus overcomes it Problems of expensive and low expression in mammalian cell lines.
  • ATIII is the major inhibitor of GzmK in human plasma in the absence of heparin
  • ATIII is the major inhibitor of GzmK in human plasma in the absence of heparin
  • the inhibition of GzmK by purified ATIII was slow, and the apparent association rate of 1.7 x 10 M * s " was of the same order of magnitude as for granzyme A.
  • inhibition of GzmK already occurred with diluted plasma samples at ineffective ATIII concentrations and was independent of ATIII, and antibodies against ATIII did not change the kinetics of inhibition in human plasma, indicating that ATIII is not the most important inhibitor of GzmK in plasma.
  • Bikunin in I ⁇ l and free bikunin inhibit the trypsin of the exocrine pancreas (36), acrosine from human sperm cells (39) and plasmin (36), but not factor Xa and plasma kallikrein
  • the blood coagulation factor Xa and plasma kallikrein are only recognized by the second Kunitz domain of Bikunin, but not by intact Bikunin and I ⁇ l for steric reasons.
  • Protease binding via the recognition loop of D2 is recognized by the amino-terminal Kunitz type domain Dl, which very likely interacts with D2-bound small trypsin-like proteases and blocks the binding of larger proteases, restricted (40) equilibrium dissociation constants for the interaction of the latter two enzymes with the isolated Kunitz domain 2 are nevertheless 20 times higher (37) than that for GzmK Until now, plasmin was the only enzyme in the interstitial and intravascular space that had a K.
  • Inhibitors of human plasma and interstitial fluids limit the extracellular effects of enzymes after their release or activation, and thus prevent undue effects on neighboring cells and distant tissues.
  • Granzymes which are controlled by liquid phase inhibitors, should act on substrates on the surface of Host cells or are present in the extracellular compartment.
  • the existence of a potent GzmK-specific Kunitz-type inhibitor in human plasma supports the idea that GzmK performs additional extracellular functions in addition to its intracellular role in the DNA fragmentation of target cells (22).
  • human Granzyme A 41, 42
  • NK natural killer cells
  • GzmK granzyme K IBs
  • inclusion bodies I ⁇ l, inter- ⁇ -trypsin inhibitor, LB, Luria-Bertani bouillon
  • RT room temperature
  • Z-Lys-SBzl N ⁇ -benzyloxycarbonyl-L-lysine-thiobenzyl ester
  • Z benzyloxycarbonyl, SBzl
  • the express ion cassettes for human GzmK precursors were cloned into the Nde I and Eco RI interfaces of pET24c (+) (Invitrogen). Transcription from the T7 promoter (black arrow) is driven by chromosomally encoded T7 RNA polymerase, which is driven by Isopropyl-1-thio-ß-D-galactopyranoside can be induced. Three constructs with amino-terminal sequence extensions (open bars) at the amino-terminus of mature GzmK (gray bar) were prepared.
  • Fig. F Preparation of catalytically active human GzmK from E. coli inclusion bodies.
  • Fig. 9 Substrate specificity of recombinant human GzmK. Enzymatic activity of unprocessed zymogen (ME pre-sequence, black bars) and activated GzmK (hatched bars) was measured using the specified thiobenzyl ester substrates at a final concentration of 0.1 nM. The enzyme concentration was 3 nM for both the unprocessed and the activated form of GzmK
  • Fig. 4 Inhibition of GzmK by purified I ⁇ l, Bikunin D2 and ATIII compared to 2.5% human plasma.
  • GzmK activity (3 nM, first to seventh column) was after incubation with 2.5% (v / v) human blood plasma (second column), 67.5 nM ATIII (third column), 26 nM Bikunin D2 (fourth column ), a mixture of 2.5 nM Bikunin D2 and 23.2 nM I ⁇ l (fifth column), 26 nM Bikunin D2 and 67.5 nM ATIII (sixth column) and with 67.5 nM ATIII, 2.5 nM Bikunin D2 and 23.2 nM I ⁇ l (seventh column) measured inhibitor concentrations were chosen so that physiological ATIII, total bikunin and total I ⁇ l concentrations of 40 times diluted human plasma were mimicked.
  • Molar concentrations of active aprotinin were determined using activity-titrated bovine trypsin CMK, chloromethyl ketone, TPCK, N-tosylphenylalanine chloromethyl ketone, TLCK, N-tosyllysine chloromethyl ketone, PMSF, phenylmethylsulfonyl fluoride

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Abstract

L'invention concerne un procédé de fabrication de sérine-protéases biologiquement actives, de domaines isolés de sérine-protéases et de leurs variantes aminoacides chez un hôte procaryote, caractérisé par addition N terminal d'une séquence auxiliaire d'un dipeptide appropriée pour la dégradation par une dipeptidylaminopeptidase, l'expression de la/ ou des sérine-protéases et/ou de leurs fragments avec des séquences auxiliaires dipeptidiques N terminal, éventuellement comme corps d'inclusion, et par renaturation de la protéine exprimée et activation de la/ ou des sérine-protéases et/ou des domaines de sérine-protéases par dédoublement de la séquence auxiliaire par une exopeptidase.
EP00964151A 1999-09-09 2000-09-08 Procede de fabrication de serine-proteases actives et de variants inactifs Withdrawn EP1214428A1 (fr)

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