WO1999010483A2 - Precurseurs de proteases activables par voie autocatalytique et leur utilisation - Google Patents

Precurseurs de proteases activables par voie autocatalytique et leur utilisation Download PDF

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Publication number
WO1999010483A2
WO1999010483A2 PCT/EP1998/005096 EP9805096W WO9910483A2 WO 1999010483 A2 WO1999010483 A2 WO 1999010483A2 EP 9805096 W EP9805096 W EP 9805096W WO 9910483 A2 WO9910483 A2 WO 9910483A2
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protease
precursor
lysc
active
inactive
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PCT/EP1998/005096
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German (de)
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WO1999010483A3 (fr
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Erhard Kopetzki
Annette Karcher
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Roche Diagnostics Gmbh
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Priority to AU93413/98A priority Critical patent/AU9341398A/en
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Publication of WO1999010483A3 publication Critical patent/WO1999010483A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • the invention relates to precursors of microbial, preferably bacterial proteases, which can be activated autocatalytically due to a protease cleavage site introduced into the molecule, and to their processes for their production and use.
  • peptide hormones such as Insulin (Jonasson, P. et al., Eur. J. Biochem. 236 (1996) 656-661; WO 96/20724), the insulin-like growth factors IGF-I and IGF-II (Nilsson, B. et al. , Methods Enzymol.
  • N-terminal initiator methionine-free growth factors and cytokines (for overview of growth factors and cytokines, see: Wang, E.A., TIBTECH 11 (1993) 379-383; Meyer-Ingold, W., TIBTECH 11 (1993) 387-392) such as e.g. IL-1-ß, IL-2, GM-CSF (Miller, CG. Et al., Proc. Natl. Acad. Sci. USA 84 (1987) 2718-2722), G-CSF (EP-B 0 513 073 ).
  • specifically cleaving proteases are e.g.
  • haemophilia B factor FX and factor VII
  • clot dissolution e.g. after heart attack (tPA, "tissue type plasminogen activator” and streptokinase) and thrombin (US Patent 5,432,062) used to promote blood clotting in wound treatment.
  • tPA heart attack
  • thrombin US Patent 5,432,062
  • proteases that selectively cleave N- or C-terminal from an amino acid, such as LysC endoprotease after lysine, or selectively from 2 specific amino acids, such as trypsin after lysine and arginine.
  • restriction proteases Carter, P .: In: Ladisch, MR; Willson, RC; Painton, CC; Builder, SE, eds., Protein Purification: From Molecular Mechanisms to Large-Scale Processes. ACS Symposium Series No. 427, American Chemical Society, pp. 181-193 (1990)
  • cleave preferably according to a recognition motif from> 2 amino acids.
  • prohormone convertases furin PC1 / PC3, PC2, PC4, PC5 and PACE4 (Perona, JJ and Craik, CS, Protein Science 4 (1995) 337-360), the blood clotting proteases FVIIa, FIXa, FXa, Thrombin, Protein C, Kallekrein, plasmin, plasminogen activator and urokinase (Furie, B. and Furie, BC, Cell 53 (1988) 505-518; Davie, EW et al., Biochem.
  • proteases such as the TEV protease (Parks, TD et al., Anal. Biochem. 216 (1994) 413-417), and / or proteases of microbial origin, such as the Kexin (Kex2p) protease from yeast (EP-A 0 467 839) and the IgA protease from Neisseria gonorrhoeae (Pohlner, J. et al., Nature 325 (1987) 458-462).
  • proteases e.g. Trypsin, carboxypeptidase B, thrombin, factor Xa, collagenase and enterokinase.
  • the methodology of the enzymatic cleavage of fusion proteins is state of the art and the proteases commercially available for cleaving fusion proteins have been described (Flaschel, E. and Friehs, K., Biotech. Adv. 11 (1993) 31-78; Sassenfeld, HM , Trends Biotechnol. 8 (1990) 88-93).
  • GB 2 083 477 describes endoproteinase LysC and a method for producing the protease from a culture broth of Lysobacter.
  • the enzymatically active endoproteinase LysC isolated from the Lysobacter culture broth has no autocatalytic cleavage site.
  • EP-A 0 134 662 and EP-A 0 137 710 describe processes for the recombinant production of calf chymosin.
  • Chymosin is a mammalian protein and not a bacterial protein and is present in an inactive pro form (prochymosin) which is autocatalytically converted at low pH values into the active pseudochymosin, from which active mature chymosin is produced.
  • the object of the invention is to provide a simple process for the recombinant production of microbial proteases as well as new autocatalytically activatable proteases and their inactive precursors.
  • the invention relates to a process for the recombinant production of a microbial protease, characterized by:
  • Proteases in the sense of the invention are to be understood as microbial, preferably bacterial, proteases, ie proteases which show the activity and characteristics of proteases which occur naturally in microorganisms, preferably in bacteria. Examples of this are given in the introduction to the description of this invention.
  • This method is particularly advantageously suitable for the recombinant production of lysyl endoproteinase (LysC).
  • Another object of the invention is an autocatalytically cleavable precursor of such a protease, which does not contain an autocatalytic cleavage site in its naturally occurring form and in which the cleavage site of the naturally occurring form is replaced by an autocatalytically cleavable cleavage site.
  • any host cell in which proteins can be formed as "inclusion bodies” is to be understood as a host cell in the sense of the invention.
  • these are prokaryotic host cells, preferably E. coli cells.
  • prokaryotic host cells preferably E. coli cells.
  • Such methods are described, for example, in FAO Marston, Biochem. J. 240 (1986) 1-12, L. Stryer, Biochemistry, WH Freeman and Company, San Francisco, 1975, pp. 24-30, TE Creighton, Progress Biophys. Molec. Biol. 33 (1978) 231-291, CH. Schein, Bio / Technology 8 (1990) 308-317, EP-B 0 114 506, A. Mitraki and J.
  • inclusion bodies are to be understood as essentially insoluble, denatured and inactive protein, which is accumulated in the cytoplasm of the host cells and which is at least partially microscopic particles.
  • Another object of the invention is accordingly a preparation of an autocatalytically cleavable precursor of a protease according to the invention, characterized in that the protein is inactive and denatured in the form of inclusion bodies in a microbial cell.
  • Another object of the invention is an aqueous solution which consists of a denaturing agent in a concentration which is suitable for dissolving the inclusion bodies and the dissolved inclusion bodies.
  • An inactive precursor of a protease is to be understood as a protein which shows no or only a very low proteolytic activity, in contrast to the active protease (at least a factor of 5, preferably a factor of 10 less activity than the active form).
  • the inactive form differs from the active form of the protease essentially in that it contains additional amino acids at which cleavage or must be cleaved to obtain the active form of the protease. These amino acids can be located at the C-terminal, N-terminal and / or within the protease.
  • a denatured and essentially inactive protein is converted into a conformation in which the protein can be activated autocatalytically and the active protease is thereby formed.
  • This conformation is usually the naturally existing conformation of the protein.
  • the sparingly soluble denatured protein is dissolved in denaturing agents, such as guanidine hydrochloride or urea, by means of processes familiar to the person skilled in the art, if necessary reduced and reduced by reducing the concentration of the denaturing agent and / or optionally by adding a denaturing aid, such as arginine, and / or a redox system, such as GSH / GSSG, converted into its active conformation.
  • denaturing agents such as guanidine hydrochloride or urea
  • An autocatalytic cleavage in the sense of the invention is to be understood as a conversion of the precursor of the protease into the active form, which takes place without the addition of further enzymes. Since the precursors of the proteases usually still have low residual teolytic activities, this is usually sufficient for the start of autocatalytic proteolysis. Preferably, the ratio of the proteolytic activity of the precursor and the active protease is 1: 5 or less.
  • Another object of the invention are autocatalytically cleavable precursors of proteases in which the cleavage site of the naturally occurring form is replaced by an autocatalytically cleavable cleavage site, and their use according to the invention.
  • proteases as an active protein, cleave the proteins of the host organism and are therefore lethal to the host organism. If the proteases are produced recombinantly in inactive form, it is necessary to convert the protein to the active form in an additional step after the recombinant production. Proteases, which are usually obtained from natural raw materials, are in turn used to cleave the zymogenic form. Such a process is therefore complex and cost-intensive.
  • the process according to the invention is characterized in that an additional step for cleaving the inactive form, by adding another protease, can be completely dispensed with.
  • this also has the advantage that the recombinant protease produced in this way is not contaminated by further proteases (in particular proteases of animal origin).
  • the inactive form of the protease can be expressed, isolated and purified during recombinant production. Such methods are known to the person skilled in the art for both eukaryotic and prokaryotic cells.
  • proteases which can be produced according to the invention are all specifically cleaving proteases, such as bacterial serine proteases, such as LysC endoproteinase or Achromobacter protease.
  • the recognition sequence and positioning of the autocatalytic cleavage site or cleavage sites inserted into the protease depend on the type of the protease and also on how the zymogenic form is activated.
  • the activation takes place by cleaving off an N- and C-terminal fragment from a prepro form during the secretion.
  • the C-terminal pro-segment should not be coded and an autocatalytic cleavage site should be inserted between the N-terminal pro-segment and the protease domain.
  • the recombinant inactive precursor of the protease differs from the naturally occurring inactive form.
  • bacterial proteases in the inactive form contain signal sequences and prosequences which are suitable for enabling the inactive protease to be removed from the cell.
  • the recombinant protease is formed in the form of insoluble inclusion bodies. It is therefore advantageous to optimize the amino acid sequence of the inactive form so that it leads as completely as possible to the formation of inclusion bodies, from which the protease can expediently be naturalized with a high yield.
  • the inclusion bodies show no enzymatic activity due to the denatured form of the protein, it is not absolutely necessary that the inactive form shows no proteolytic activity at all. It is even preferred that the inactive form show low proteolytic activity in order to accelerate the autocatalytic cleavage after naturation.
  • the cleavage site or cleavage sites are preferably inserted while maintaining the native primary structure (protease domain). According to the invention, it is thus possible, for example, to produce endoproteinase LysC with a natural sequence in a simple manner recombinantly in prokaryotes without the protein N-terminally starting with the amino acid methionine encoded by the start codon.
  • the active protease produced according to the invention is immobilized.
  • immobilization can be carried out, for example, by binding to a polymeric, insoluble carrier or by self-crosslinking (see, for example, US Pat. No. 4,634,671 and EP-A 0 367 302).
  • the inactive form can essentially (except for the autocatalytic cleavage site) correspond to the natural inactive form of the protease.
  • the sequence of the fragment or fragments to be cleaved is further modified. Suitable autocatalytic cleavage sites for preferred enzymes are given in the table below: Enzymatic cleavage (restriction endoprotease)
  • lysyl endoproteinase LysC from Lysobacter enzymogenes are preferred.
  • the lysyl endoproteinase LysC (E.C 3.4.21.50) is a secreted proteinase that cleaves specifically at the C-terminal of the amino acid lysine. It is obtained from the culture filtrate of microorganisms of the order Lysobacterales (preferably from Lysobacter enzymogenes) using conventional enzyme purification methods (US Pat. No. 4,414,332).
  • the LysC endoproteinase consists of a polypeptide chain (monomer) with an apparent molecular weight of approx. 30 kDa and belongs to the family of serine proteases.
  • the content of the LysC endoproteinase secreted into the medium is low, the purification is complex and the enzyme is therefore very expensive.
  • LysC endoproteinase in biotechnology (eg for the recombinant production of lysine-free peptides such as natriuretic peptides (WO 97/11186) and hirudin derivatives (EP-A 0 667 355) by LysC-mediated release of the desired peptide from a fusion protein and / or
  • the enzymatic semisynthesis of human insulin and insulin analogs (Morihara, K., TIBTECH 5 (1987) 164-170; EP-B 0 017 938; WO 83/02772; EP-B 0 387 646) has hitherto been very limited due to the limited availability and a non-economical production of the LysC endoproteinase.
  • the invention accordingly relates to a lysyl endoproteinase (LysC) which is completely free from proteins which are found naturally as contaminants of the protease mentioned when isolated from natural sources.
  • the sequence of the preferred proteinase is completely identical to the sequence of the naturally occurring protease.
  • the nucleic acid sequence coding for LysC endoproteinase from Lysobacter enzymogenes and the amino acid sequence are not yet known and are the subject of the invention. dung.
  • the invention also relates to processes for the recombinant production of LysC endoproteinase, expression vectors and host cells which have been transformed with the expression vectors.
  • LysC endoproteinase corresponds to the published sequence of the lysyl endopeptidase from Achromobacter lyticus (Ohara, T. et al, J. Biol. Chem. 264 (1989) 20625-20631; Sakiyama, F. et al, EP- B 0 387 646) has a degree of homology (amino acid sequence identity) of 77%.
  • the amino acid sequences differ in 61 from 268 amino acid residues.
  • A. lyticus protease I Likewise preferred are autocatalytically cleavable inactive precursors of the Achromobacter lyticus protease I (API).
  • API Achromobacter lyticus protease I
  • the cloning and sequencing of the A. lyticus protease I gene is published (Tsunasawa, S. et al., J. Biol. Chem. 264 (1989) 3832-3839; Ohara, T. et al., J. Biol. Chem. 264 (1989) 20625-20631; Sakiyama, F. et al., EP-B 0 387 646).
  • the amino acid sequence derived from the A. lyticus Protease I gene codes for a polypeptide chain of 653 amino acids.
  • N-terminal prepro segment including a 20 amino acid residue long signal sequence and ends with a 180 amino acid residue long serine / threonine-rich C-terminal prosegment.
  • the mature active 268 amino acid residues A. lyticus protease I is formed during the secretion by enzymatic cleavage of the N- and C-terminal prosegments with the formation of 3 disulfide bridges.
  • a process for the secretion of proteins and peptides into the medium of bacteria which have an inner and outer cell wall is claimed by Woldike, F. and Hastrup, S. (WO 96/17943) using the prepro segment of the A. lyticus Protease I.
  • the expression / secretion of small amounts of natively processed API into the medium of E. coli is claimed by Woldike, F. and Hastrup, S. (WO 96/17943) using the prepro segment of the A. lyticus Protease I.
  • the expression / secretion of small amounts of natively processed API into the medium of E. coli immunological detection with anti-API antibodies, exact information on the yield are missing
  • a shortened API variant gene deletion of the region coding for the C-terminal prosegement
  • the LysC endoproteinase belongs to the serine protease family.
  • the activation of the LysC endoproteinase is very likely to take place like the Lysyl endoproteinase from Achromobacter lyticus during the secretion by cleavage of an N-terminal prosegment.
  • SEQ ID NO: l the amino acid sequence of Lysyl endoproteinase (LysC) from Lysobacter enzymogenes.
  • SEQ ID NO: 2 the nucleotide sequence of the synthetically produced lysyl endoproteinase variant gene.
  • the protein concentration of the proteases and protease variants was determined by determining the optical density (OD) at 280 nm using the molar extinction coefficient calculated on the basis of the amino acid sequence.
  • the vector for the expression of the autocatalytically activatable proteases is based on the
  • Expression vector pSAM-CORE for core streptavidin.
  • the preparation and description of the plasmid pSAM-CORE is described in WO 93/09144 by Kopetzki, E. et al. described.
  • the core streptavidin gene was replaced by the desired protease gene in the pSAM-CORE vector.
  • the amino acid sequence of the Lysobacter enzymogenic endoproteinase LysC (Ka No. 1420429) commercially available from Boehringer Mannheim GmbH (Mannheim, Germany) was determined according to the prior art by overlapping peptide sequencing.
  • the denatured LysC polypeptide chain was cleaved enzymatically with trypsin (hydrolysis according to the basic amino acids Lys and Arg) and chemically using cyanogen bromide (hydrolysis according to the amino acid methionine).
  • the peptide mixtures were fractionated by RP HPLC and the amino acid sequence of each peptide was determined according to the sequential Edman method (Hunkapiller, MW et al., Methods Enzymol.
  • the complete amino acid sequence of the LysC polypeptide chain was determined by combining the overlap of the individual peptides.
  • the determined LysC endoproteinase amino acid sequence is shown in SEQ ID NO: 1.
  • the LysC endoproteinase from Lysobacter enzymogenes has a homology (amino acid sequence identity) of 77% to the Lysyl endoproteinase from Achromobacter lyticus.
  • the determined Lysobacter enzymogenic LysC protein sequence was used in the construction of the synthetic LysC-MGSK gene.
  • the "codon usage” was adapted to the codons used with preference in E. coli.
  • suitable unique restriction endonuclease interfaces were introduced within the coding region and at the ends of the LysC-MGSK gene with regard to the construction of LysC variant genes and the recloning of the LysC expression cassette.
  • the LysC-MGSK gene was produced by Genosys (Genosys Biotechnologies, Inc. Cambridge, England) by chemical synthesis from oligonucleotides.
  • the double-stranded LysC-MGSK gene (FIG. 1) was constructed by "annealing” and ligation of the oligonucleotides and then into the EcoRI and HindIII interface of the E. coli standard vector pGEM-3Zf from Promega (Promega Corp., Madison, WI, USA) ) cloned.
  • the predetermined DNA sequence of the cloned LysC-MSGK gene was confirmed by DNA sequencing.
  • the vector for the expression of the LysC-MGSK endoproteinase is based on the expression vector pSAM-CORE for core streptavidin.
  • the core streptavidin gene was replaced by the desired LysC-MGSK gene in the pSAM-CORE vector.
  • the plasmid pGEM-3ZF-LysC-MGSK was digested with the restriction endonucleases Ncol and Hindill and the approx. 830 bp NcoI / Hindlll-LysC-MGSK fragment after purification by agarose gel electrophoresis into the approx. 2.55 kBp long NcoI / Hindlll- pSAM- Ligated CORE vector fragment.
  • the desired plasmid pLysC-MGSK was identified by restriction mapping and the LysC-MSGK gene was checked by DNA sequencing.
  • LysC-MGSK variant gene an E. coli K12 strain (e.g. UT5600; Grodberg, J. and Dünn, JJ, J. Bacteriol. 170 (1988) 1245-1253) was used with the expression plasmid pLysC-MGSK (example 3) Ampicillin resistance) and the lacl q repressor plasmid pUBS520 (kanamycin resistance, preparation and description see: Brinkmann, U. et al., Gene 85 (1989) 109-114).
  • the transformed UT5600 / pUBS520 / pLysC-MGSK cells were shaken in DYT medium (1% (w / v) yeast extract, 1% (w / v) Bacto Tryptone, Difco, and 0.5% NaCl) with 50-100 mg / 1 ampicillin and 50 mg / 1 kanamycin at 37 ° C to an optical density at 550 nm (OD 550 ) of 0.6-0.9 and then induced with IPTG (1-5 mmol / 1 final concentration).
  • DYT medium 1% (w / v) yeast extract, 1% (w / v) Bacto Tryptone, Difco, and 0.5% NaCl
  • the cells were harvested by centrifugation (Sorvall RC-5B centrifuge, GS3 rotor, 6000 rpm, 15 min), with 50 mmol / 1 Tris-HCl buffer, pH 7.2 washed and stored at -20 ° C until further processing.
  • the cell yield from a 1 l shake culture was 4-5 g (wet weight).
  • LysC-MGSK expression in the transformed UT5600 / pUBS520 / pLysC-MGSK cells was analyzed.
  • cell pellets were resuspended from 1 ml of centrifuged growth medium in 0.25 ml of 10 mmol / 1 Tris-HCl, pH 7.2, and the cells were sonicated (2 pulses of 30 s with 50% intensity) using a Sonifier Cell Disruptor B15 Branson company (Heusenstamm, Germany) open minded.
  • the insoluble cell components were sedimented (Eppendorf 5415 centrifuge, 14000 rpm, 5 min) and the supernatant with 1/5 volume (vol) 5xSDS sample buffer (IxSDS sample buffer: 50 mmol 1 Tris-HCl, pH 6.8, 1% SDS , 1% mercaptoethanol, 10% glycerin, 0.001% bromophenol blue) was added.
  • the insoluble cell debris fraction (pellet) was resuspended in 0.3 ml IxSDS sample buffer with 6-8 M urea, the samples were incubated for 5 min at 95 ° C. and centrifuged again. After that, the proteins were gel electrophoresis by SDS-polyacrylamide (PAGE) separated (Laemmli, UK, Narure 227 (1970) 680-685) and stained with Coomassie Brilliant Blue R dye.
  • the LysC-MGSK endoproteinase synthesized in E. coli was homogeneous and was found exclusively in the insoluble cell debris fraction ("inclusion bodies", IBs). The level of expression was 10-50%) based on the total E. coli protein.
  • the cell pellet from 3 1 shake culture (approx. 15 g wet weight) was resuspended in 75 ml 50 mmol / 1 Tris-HCl, pH 7.2.
  • the suspension was mixed with 0.25 mg / ml lysozyme and incubated at 0 ° C for 30 min.
  • the cells were disrupted mechanically by means of high-pressure dispersion in a French press from SLM Amico (Urbana, IL, USA) .
  • the DNA was then digested for 30 min at room temperature (RT).
  • the purified IBs were at a concentration of 100 mg IB pellet (wet weight) / ml corresponding to 5-10 mg / ml protein in 6 mol / 1 guanidinium HCl, 100 mmol 1 Tris-HCl, 20 mmol / 1 EDTA, 150 mmol 1 GSSG and 15 mmol / 1 GSH, pH 8.0 dissolved with stirring at RT in 1-3 hours.
  • the pH was then adjusted to pH 5.0 and the insoluble constituents were separated off by centrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 16000 rpm, 10 min). The supernatant was dialyzed against 100 vol. 4-6 mol / 1 guanidinium-HCl pH 5.0 for 24 hours at 4 ° C. c) Naturation
  • the clear proteinase-containing supernatant was concentrated 10-15-fold by cross-flow filtration in a Minisette (membrane type: Omega 3K) from Filtron (Karlstein, Germany) and to remove guanidinium-HCl and arginine against 100 vol. 20 mmol / 1 Tris-HCl, pH 9.0, dialyzed for 12-24 hours at 20-25 ° C.
  • Minisette membrane type: Omega 3K
  • Precipitated protein was separated by centrifugation (Sorvall RC-5B centrifuge, SS34 rotor, 16000 rpm, 20 min) and the clear supernatant with a Nalgene ® single-use filtration unit (pore diameter: 0.2 mm) from Nalge (Rochester, NY, USA) ) filtered.
  • the active LysC endoproteinase from the naturalization batch can be further purified if necessary using chromatographic methods known to the person skilled in the art.
  • the bound material was eluted by a gradient of 50-1000 mmol / 1 NaCl in 20 mmol / 1 Tris-HCl, pH 9.0 (2 SV / hour).
  • the proteinase-containing fractions were identified by non-reducing and reducing SDS PAGE and the elution peak was pooled.
  • the concentrated naturation mixture dialyzed against 20 mmol / 1 Tris-HCl, pH 9.0, was placed on a benzamidine-Sepharose-CL-6B column equilibrated with the same buffer (1.0 x 10 cm, V 8 ml; loading capacity: 2-3 mg protein / ml gel) from Pharmacia Biotech (Freiburg, Germany) (2 SV / hour) and washed with the equilibration buffer until the absorption of the eluate at 280 nm reached the blank value of the buffer.
  • the bound material was eluted by 10 mmol / 1 benzamidine in 20 mmol / 1 Tris-HCl and 200 mmol 1 NaCl, pH 9.0 (2 SV / hour).
  • the protease-containing fractions were identified by non-reducing and reducing SDS PAGE and activity determination.
  • the serine protease inhibitor benzamidine used for elution was removed by dialysis against 1 mmol / 1 HC1.
  • the activity determination of LysC was carried out in a volume of 1 ml in 25 mmol 1 Tris-HCl, 1 mmol 1 EDTA, 0.1% PEG 8000, pH 8.5 and 0.1 mmol / 1 of the chromogenic substrate Chromozym ® PL (Tosyl -Gly-Pro-Lys-4-pNA, cat.no.838250, Boehringer Mannheim GmbH, Mannheim, Germany) at 25 ° C.
  • 1 Chromozym ® PL Unit LysC is defined as the amount of enzyme that releases 1 ⁇ mol pNitroaniline (pNA) per minute at 25 ° C from the chromozymic substrate. The results are
  • Bristow, A.F . The current status of therapeutic peptides and proteins. In: Hider, R.C .;

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Abstract

L'invention concerne un procédé de préparation recombinée de protéase microbienne, qui se caractérise par: a) transformation d'une cellule hôte avec un acide nucléique recombiné, qui code un précurseur inactif d'une protéase microbienne, identifié par la forme active de ladite protéase et est coupé de manière spécifique pour donner lieu à la protéase active; b) mise en culture de la cellule hôte de manière que le précurseur de la protéase figure sous forme de corps d'inclusion dans la cellule hôte; c) par isolement des corps d'inclusion et par retour à l'état naturel dans des conditions où la partie de protéase du précurseur apparaît dans sa conformation naturelle et d) par séparation par voie autocatalytique du précurseur revenu à l'état naturel, pour donner lieu à la protéase active.
PCT/EP1998/005096 1997-08-22 1998-08-12 Precurseurs de proteases activables par voie autocatalytique et leur utilisation WO1999010483A2 (fr)

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EP97119405.5 1997-11-06

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6159722A (en) * 1997-12-03 2000-12-12 Boehringer Mannheim Gmbh Chimeric serine proteases
WO2001011057A1 (fr) * 1999-08-09 2001-02-15 Biochemie Gesellschaft M.B.H. Production de proteines
WO2001011056A1 (fr) * 1999-08-09 2001-02-15 Biochemie Gesellschaft M.B.H. Production de proteines par clivage autoproteolytique
WO2006113959A2 (fr) * 2005-04-26 2006-11-02 Sandoz Ag Composes organiques

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2083477A (en) * 1980-09-10 1982-03-24 Boehringer Mannheim Gmbh Endoproteinase-lys-c and process for the preparation thereof
EP0114506A1 (fr) * 1982-12-22 1984-08-01 Genentech, Inc. Méthodes de purification et réactivation des protéines hétérologues précipitées
EP0134662A1 (fr) * 1983-07-07 1985-03-20 Genex Corporation Production de rénine de veau bovin
EP0137710A2 (fr) * 1983-09-01 1985-04-17 Genex Corporation Procédé de récupération et d'activation de la prochymosine de veau produite micro-biologiquement
DE4140699A1 (de) * 1991-01-11 1992-07-16 Boehringer Mannheim Gmbh Rekombinante iga-protease

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2083477A (en) * 1980-09-10 1982-03-24 Boehringer Mannheim Gmbh Endoproteinase-lys-c and process for the preparation thereof
EP0114506A1 (fr) * 1982-12-22 1984-08-01 Genentech, Inc. Méthodes de purification et réactivation des protéines hétérologues précipitées
EP0134662A1 (fr) * 1983-07-07 1985-03-20 Genex Corporation Production de rénine de veau bovin
EP0137710A2 (fr) * 1983-09-01 1985-04-17 Genex Corporation Procédé de récupération et d'activation de la prochymosine de veau produite micro-biologiquement
DE4140699A1 (de) * 1991-01-11 1992-07-16 Boehringer Mannheim Gmbh Rekombinante iga-protease

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Y.E. CHENG ET AL: "HIGH-LEVEL SYNTHESIS OF RECOMBINANT HIV-1 PROTEASE AND THE RECOVERY OF ACTIVE ENZYME FROM INCLUSION BODIES" GENE, Bd. 87, Nr. 2, 15. M{rz 1990, Seiten 243-248, XP000114192 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6660492B1 (en) 1997-12-03 2003-12-09 Boehringer Mannheim Gmbh Chimeric serine proteases
US6171842B1 (en) 1997-12-03 2001-01-09 Boehringer Mannheim Gmbh Chimeric serine proteases
US6159722A (en) * 1997-12-03 2000-12-12 Boehringer Mannheim Gmbh Chimeric serine proteases
CZ302366B6 (cs) * 1999-08-09 2011-04-13 Sandoz Ag Zpusob rekombinantní produkce heterologního polypeptidu pomocí peptidu s autoproteolytickou aktivitou
WO2001011056A1 (fr) * 1999-08-09 2001-02-15 Biochemie Gesellschaft M.B.H. Production de proteines par clivage autoproteolytique
US6936455B1 (en) 1999-08-09 2005-08-30 Sandoz Ag Production of heterologous proteins using an Npro autoprotease of a pestivirus and inclusion bodies
US7378512B2 (en) 1999-08-09 2008-05-27 Biochemie Gesellschaft M.B.H Production of proteins by autoproteolytic cleavage
WO2001011057A1 (fr) * 1999-08-09 2001-02-15 Biochemie Gesellschaft M.B.H. Production de proteines
WO2006113959A2 (fr) * 2005-04-26 2006-11-02 Sandoz Ag Composes organiques
WO2006113959A3 (fr) * 2005-04-26 2007-03-22 Sandoz Ag Composes organiques
EP2348053A3 (fr) * 2005-04-26 2011-11-02 Sandoz AG Ligands oligopeptidiques
US8163890B2 (en) 2005-04-26 2012-04-24 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein
US8372959B2 (en) 2005-04-26 2013-02-12 Sandoz Ag Production of recombinant proteins by autoproteolytic cleavage of a fusion protein

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AR012265A1 (es) 2000-09-27
AU9341398A (en) 1999-03-16

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