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WO2012104099A1 - Procédé de production de trypsine recombinante - Google Patents

Procédé de production de trypsine recombinante Download PDF

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Publication number
WO2012104099A1
WO2012104099A1 PCT/EP2012/000497 EP2012000497W WO2012104099A1 WO 2012104099 A1 WO2012104099 A1 WO 2012104099A1 EP 2012000497 W EP2012000497 W EP 2012000497W WO 2012104099 A1 WO2012104099 A1 WO 2012104099A1
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trypsinogen
trypsin
protein
recombinant
anyone
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PCT/EP2012/000497
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English (en)
Inventor
Peter König
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Glucometrix Ag
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Publication of WO2012104099A1 publication Critical patent/WO2012104099A1/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)
    • C12N9/64—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)
    • C12N9/6427—Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag

Definitions

  • the invention relates to a process for the production of recombinant trypsin and its use for processing of an insulin precursor into insulin.
  • the invention relates to a process for the pro- duction of recombinant trypsin starting from recombinant trypsinogen produced in a prokaryotic host cell.
  • the recombinant trypsinogen is produced by the host cell in form of inclusion bodies.
  • the trypsinogen contained in the inclusion bodies is purified and subsequently refolded into its native conformation involving formation of disulfide bridges.
  • the refolded trypsinogen is then further processed into active trypsin.
  • Trypsin is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins. It cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. Trypsin is produced in the pancreas as the inactive proenzyme trypsinogen, which is activated by enteropeptidase to form trypsin. Once activated, the trypsin can activate more trypsinogen into trypsin.
  • Trypsin is used for numerous biotechnological processes such as for the production of insulin, where it is used in combination with carboxypeptidase B for processing of an insulin precursor such as human proinsulin into active insulin.
  • carboxypeptidase B for processing of an insulin precursor such as human proinsulin into active insulin.
  • the C-peptide or any other peptide located between the A- and B-chains of insulin is cleaved off by action of trypsin. Due to the high demand for insulin as medicament, e.g. for the treatment of diabetic disorders worldwide, there is also a high demand for trypsin for use in the production of insulin.
  • Trypsin is commercially available as product purified from bovine or pig pancreas or as recombinant product manufactured by biotechnological processes.
  • Gene sources are generally bovine, rat or human trypsin. Purification from animal sources such as animal pancreas, however, bares the risk of contamination of the trypsin product with pathogenic agents such as viruses or priones. It therefore requires excessive purification of the trypsin product making the processes costly and economically less valuable. Thus, these products are usually not useful for the manufacture of pharmaceutically agents such as insulin.
  • biotechnological ly manufactured recombinant trypsin provides for a constant and reproductive quality of the product. The recombinant production of trypsin has been described in two mainly applied expression systems.
  • the first system uses expression in yeast such as Saccharomyces cerevisiae (S. cerevisiae) or Pichia pastoris (P. pastoris).
  • yeast such as Saccharomyces cerevisiae (S. cerevisiae) or Pichia pastoris (P. pastoris).
  • EP 1 399 568 B l describes a method for the recombinant production of trypsin in a host such as Saccharomyces or Pichia, wherein the expression product is secreted into the culture medium and then purified from the culture medium.
  • trypsin by expression of trypsinogen in yeast generally results in low and economically insufficient yields.
  • the second and more favourable expression system is the recombinant expression in prokaryotic organisms (synonymously "prokaryotic host” or “prokaryotic host cell”) such as Escherichia coli (E. coli). Due to high rate of synthesis and rapid growth, the primary source for the manufacturing of biosynthetic recombinant proteins such as trypsin in yields that are economically reasonable is its production in E. coli. However, manufacturing of recombinant trypsin in a prokaryotic organism is often also accompanied by several disadvantages.
  • inclusion bodies Upon expression in the host cell high molecu- lar weight aggregates are formed, often referred to as "inclusion bodies", which result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment.
  • inclusion bodies Upon expression in the host cell high molecu- lar weight aggregates are formed, often referred to as "inclusion bodies", which result from the inability of the expressed proteins to fold correctly in an unnatural cellular environment.
  • the protein is present in the insoluble inclusion bodies in denatured form, therefore requiring the use of detergents and denaturants to isolate and solubilize the protein.
  • the isolated protein must subsequently be refolded in vitro into the native conformation. This also requires the formation of the correct disulfide bridges (also synonymously referred to as cystine bridges), which were usually not formed intracellularly.
  • the disulfide bridges are generally essential in maintaining the native conformation and biological activity of the protein molecule.
  • renaturation it is necessary to treat the isolated recombinant protein in vitro under conditions that allow extracellular formation of disulfide bridges and folding of the protein into its native conformation. This process is generally called renaturation.
  • renaturation of recombinant protein is a difficult and laborious process and often leads to unsatisfactory results and low yields caused by the formation of incorrect disulfide bridges and insoluble protein aggregates.
  • the incorrect formation of disulfide bridges prevents the recombinant protein to fold into its native conformation.
  • Such incorrectly folded protein cannot be converted or processed into its active form, and in consequence results in decreased yield of active product.
  • the technical problem of the present invention is to provide an improved process for the production of recombinant trypsin.
  • the technical problem is to provide improved processes for the production of recombinant trypsin starting from trypsinogen isolated from inclusion bodies after expression in a prokaryotic host.
  • the inventors of the present invention have surprisingly found that the above described technical problems can be overcome by providing a process for the production of recombinant trypsin, in which recombinant trypsinogen is isolated from inclusion bodies, subsequently purified and the purified protein is then subjected to a renaturation process before processing trypsinogen into trypsin to yield the active product. It could surprisingly be shown by the present invention that purified trypsinogen isolated from inclusion bodies can be effectively renatured leading to higher yields of correctly folded product compared to recombinant trypsinogen subjected to a renaturation process without the prior purification step.
  • the present invention provides a process for the production of recombinant trypsin comprising the following steps a) to g) of a) transforming a prokaryotic host cell with a nucleic acid which codes for trypsinogen,
  • the recombinant trypsin produced by the process of the subject invention can be used for processing of an insulin precursor, preferably a human insulin precursor such as human proinsulin, for the production of insulin.
  • heterologous expression means that the protein is experiment- ally put into a cell that does not normally make (i.e., express) that protein.
  • Heterologous polypeptide or heterologous protein thus refers to the fact that the transferred DNA coding for a polypeptide or protein such as trypsinogen was initially cloned from or derived from a different cell type or a different species from the recipient.
  • the gene encoding trypsinogen can be made synthetically and then transferred into the host organism, which as native organism does not pro- Jerusalem that polypeptide or protein. Therefore, the genetic material encoding for the polypeptide or protein can be added to the recipient cell by recombinant cloning techniques.
  • the genetic material that is transferred for the heterologous expression should be within a format that encourages the recipient cell to express the recombinant DNA as open reading frame (O F) to synthesize a protein, i.e., it is put in an expression vector.
  • O F open reading frame
  • polypeptide refers to a single linear chain of amino acids.
  • protein refers to a polypeptide, which has the ability to form into a specific conformation. In the context of the present invention the terms polypeptide and protein can generally be used interchangeably for polypeptides of a specific length.
  • recombinant DNA refers to the form of artificial DNA such as a synthetic DNA or cDNA, e.g. coding for trypsinogen that is created through the introduction of the DNA into an organism such as E. coli for the purpose of expression of the polypeptide or protein encoded by the recombinant DNA.
  • a "recombinant protein” thus is a protein that is derived from the recombinant DNA by expression of the recombinant DNA in the host cell.
  • a “correctly folded” protein such as native trypsinogen or native trypsin refers to a molecule, which has the three dimensional conformation and disulfide bridges as found in the naturally occurring, biologically active protein.
  • a prokaryotic host cell is transformed with a nucleic acid which codes for trypsinogen.
  • the host cell can be transformed with a nucleic acid, which codes for an inactive trypsin precursor (zymogen), including derivatives or homologues of trypsinogen or any trypsin precursor that can be processed into an enzymatically active trypsin product.
  • Trypsinogen preferably refers to human trypsinogen.
  • the amino acid sequence of human trypsinogen is deposited under accession number AAA61232.
  • trypsinogen can be selected from any mammalian trypsinogen such as bovine trypsinogen, rat trypsinogen or pig trypsinogen.
  • the nucleic acid codes for a protein comprising, from N- terminus to C-terminus, methionine as start codon and amino acids 13 to 247 of the human trypsin zymogen (synonymously: trypsinogen), enumeration of amino acids according to Uni- ProtKB/Swiss-Prot P07477.
  • trypsinogen enumeration of amino acids according to Uni- ProtKB/Swiss-Prot P07477.
  • the DNA coding for trypsinogen is thus incorporated by standard cloning techniques into an expression vector.
  • the expression vector provides all elements necessary for recombinant expression of trypsinogen in the heterologous host.
  • Suitable expression vectors are commercially available and include standard expression vectors for expression in E. coli such as pQET7 available from Qiagen in which the gene encoding the recombinant protein is expressed under control of the T7 promoter. Transformation of the host cell by the expression vector can be achieved as described by Sambrook et al., Cold Spring Harbor Laboratory Press, 1998.
  • Suitable E. coli strains are commercially available and include various strains derived from E. coli BL21 such as E. coli C41 available from Lucigen, preferably E. coli C41(DE3).
  • the prokaryotic host which preferably is an E. coli cell, is thus transformed by a DNA coding for trypsinogen. Transformation can be achieved by standard cloning techniques such as transformation of electro competent cells or chemically made competent cells.
  • the amino acid sequence of trypsinogen may further comprise or be fused to a sequence, which promotes the purification of the recombinant protein.
  • a sequence, which is preferably used for purification of a recombinant protein such as recombinant trypsinogen, is a His-tag.
  • His- tag or polyhistidine-tag is an amino acid motif in proteins that consists of at least four histidine (His) residues.
  • His-tag consists of six histidine residues and is thus also known as hexa histidine-tag or 6xHis-tag.
  • His-tag can be used for affinity purification of the tagged recombinant protein, e.g. after expression in E. coli.
  • Various purification kits for histidine- tagged proteins are available from Qiagen, Sigma, Thermo Scientific, GE Healthcare, Macherey- Nagel and others.
  • the codon usage of the nucleotide sequence according to the present invention is adapted for the expression in the respective organism (E.L. Winnacker, Gene und Klone, Verlag Chemie, 1985, 224-241 , Codon usage tabulated from the international DNA sequence databases: status for the year 2000; Nakamura et al., 2000, Nucl. Acids Res. 28, 292).
  • the DNA coding for trypsinogen is preferably adapted for expression in the specific organism.
  • the encoding DNA can be adapted according to the codon usage of e.g. E. coli.
  • the inventors have also achieved high yields of trypsinogen expression when using a nucleic acid that is optimized for expression in Pichia pastoris and expressing this nucleic acid in E. coli.
  • the invention also refers to a process in which the trypsinogen coding sequence is adapted for expression in yeast such as P. pastoris and which is used for expression of trypsinogen in a prokaryotic host such as E. coli.
  • the DNA coding for trypsinogen may be obtained by standard procedures such as cloned DNA, e.g., a DNA "library”, by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof, purified from the desired cell.
  • Expression from certain promoters contained in the expression vector can be elevated in the presence of certain inducers; thus, expression of the recombinant protein may be controlled in order to optimize expression yields.
  • a preferred expression system is under control of the T7 promoter of E. coli and induced by the presence of IPTG (isopropyl ⁇ -D-l-thiogalactopyranoside).
  • step b) the recombinant cell resulting from step a) is cultivated under conditions that allow expression of the recombinant nucleic acid by formation of inclusion bodies, i.e. the nucleic acid is recombinantly expressed by the cell.
  • the resulting recombinant prokaryotic host is typically cultivated in a suitable medium.
  • suitable liquid media for growing the host organism include synthetic media, full or half media.
  • coli include Luria Broth (LB), 2xYT or, in a particularly preferred embodiment of this invention, a fully synthetic medium based on a phosphate buffer, a nitrogen source like ammonium chloride, a carbon- and energy source like glucose or glycerol, trace elements, and an amino acid supplement to enhance growth ( orz et al., 1994, J. Biotech. 39, 59).
  • Suitable conditions for cultivation are adapted to the organism according to standard procedures. These include inocculation of the growth media with a starter culture and incubating the cells at a temperature of generally between 20 °C and 42 °C. When grown in a flask, the concentration of dissolved oxygen can be enhanced by rigorous shaking. In a bioreactor, ample air supply is generally necessary.
  • the pH of the culture should be kept between 5 and 8.
  • expression of the recombinant protein can be induced by typical inducers such as by addition of isopropyl-B-D-galactopyranosid (IPTG) or lactose.
  • IPTG isopropyl-B-D-galactopyranosid
  • lactose lactose
  • the cells are usually harvested e.g. by filtration or centrifugation and then disintegrated to further isolate the recombinant protein form the broken cells.
  • Disintegration of cells can be achieved by high pressure homogenisation using a high pressure cell such as a french press cell. Other methods of disintegration of cells include enzymatic treatment with lysozyme and/or sonication.
  • the prokaryotic host such as E. coli the recombinant trypsinogen is usually present in form of insoluble inclusion bodies.
  • the inclusion bodies have to be isolated from the broken cells. Isolation of inclusion bodies can be achieved washing the broken cells with mild detergents, e.g. Tween 20 or Triton X 100 and/or with low concentrations of urea, preferably up to 2 M.
  • step d) the isolated inclusion bodies are solubilized to solve the recombinant trypsinogen contained in the inclusion bodies.
  • Solubilization can be achieved by using a strong denaturant such as urea, which preferably is used in a concentration of 6 to 8.5 M, more preferably of 7 to 8 M.
  • a strong denaturant such as urea
  • guanidine hydrochloride can be used as denaturant in a concentration of 6 to 9 M, prefer- ably between 7 and 8.5 M.
  • the solubilization buffer should contain an agent that reduces disulfide bridges in proteins, such as DTT or ⁇ -mercaptoethanol.
  • concentrations for DTT are between 5 to 50 mM, preferably between 10 to 20 mM, and for ⁇ - mercaptoethanol between 10 mM and 200 mM, most preferably between 25 and 75 mM. Solubilization of inclusion bodies can also be achieved as described in Peterson et al., 2001 , Biochem- istry, 40, 6275.
  • the protein concentration used in step d) is preferably between 0.25 and 50 mg/ml, more preferably between 0.5 and 10 mg/ml, most preferably between 0.75 and 5 mg/ml.
  • the inclusion bodies can be incubated in a buffer as described above for 20 to 180 min, preferably between 30 and 120 min, most preferably between 45 and 90 min. Typically, incubation is performed under mild shaking or mixing. After incubation, the solution containing the solubilized trypsinogen is generally isolated from the non-solubilized material by centrifugation or dialysis, preferably by centrifuga- tion.
  • the purity of trypsinogen prior to purification of the solubilized protein resulting from step d) is preferably at least 25 wt.%, more preferably between 30 and 70 wt.%, based on the total protein.
  • step e) the solubilized trypsinogen is subjected to one or more purification steps.
  • Purification is preferably performed under denaturing conditions.
  • Purification can be performed by one or more purification methods such as dialysis, cationic chromatography, anionic chromatography, reversed phase chromatography, affinity chromatography and size exclusion chromatography as long as they are compatible with buffers in which the trypsinogen can be kept in the denatured state. This is achieved by using purification buffers which contain a strong denaturant such as urea in a concentration of 7 to 8 M or guanidine hydrochloride in a concentration of 6 to 9 M. In case of cationic or anionic chromatography, urea is preferred as denaturant.
  • the proteins are kept in a reduced state by including a DTT at a concentration range of 1 to 20 mM, most preferably about 5 mM, or ⁇ -mercaptoethanol at a concentration range of 5 to 20 mM, most preferably about 10 mM.
  • Preferred purification methods are cationic chromatography and size exclusion chromatography, most preferably cationic chromatography.
  • inclusion bodies consist mostly of the target protein. However, they may also contain varying amounts of contaminating polypeptides (5 to 50 wt.%), phospholipids (0.5 to 13 wt.%) as well as residual amounts of nucleic acids (Valax and Georgiou, 1993, Biotechnol. Prog., 9, 539). It could be shown by the inventors that purified trypsinogen can be renatured at a much higher rate in comparison to non-purified trypsinogen under similar renaturing conditions and using similar trypsinogen concentrations in the renaturation buffer. This significantly higher renaturation rate for the purified material results in an overall higher yield of active trypsinogen obtained from inclusion bodies.
  • the purity of trypsinogen resulting from purification in step e) is preferably at least 50 wt.%, more preferably between 70 and 99 wt.%, most preferably between 90 and 99 wt.%, based on the total protein.
  • the concentration of trypsinogen after purification in step e) of the solubilized inclusion bodies is preferably from 0.5 to 50 mg/ml, even more preferably from 5 to 10 mg/ml.
  • the purified trypsinogen is subjected to a renaturation process under conditions that allow refolding of trypsinogen into its native three-dimensional conformation and forming of correct disulfide bonds.
  • Suitable conditions include the choice of an appropriate buffer and an appropriate pH for isolation and refolding of the recombinant protein as described by De Bernardez Clark et al., 2001 , Current Opinion in Biotechnology, 12, 202 and Middelberg et al., 2002, Trends in Biotechnology, 20, 437.
  • the renaturation buffer usually contains a buffering agent such as HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) or Tris HCl (hydrochloric acid salt of tris(hydroxymethyl) ami- nomethane), preferably Tris HCl, for maintaining a constant pH.
  • a buffering agent such as HEPES (4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid) or Tris HCl (hydrochloric acid salt of tris(hydroxymethyl) ami- nomethane), preferably Tris HCl, for maintaining a constant pH.
  • the pH is usually kept constant in a range of pH 7.5 to 9.5, more preferably of pH 8 to 8.5.
  • Tris HC1 can be used in a concentration of 5 to 500 mM, more preferably 10 to 100 mM, most preferably 20 to 50 mM.
  • a suitable pair of reduced and oxidized low molecular weight thiol reagents is usually included in the renaturation buffer to facilitate the exchange of disulfide bridges, thereby increasing the rate at which the correct disulfide bridges within the protein are formed.
  • Such pairs can be reduced/oxidized glutathione, reduced/oxidized mercaptoethanol, cysteine/cystine in a total concentration of 0.1 mM to 10 mM, preferably 1 to 5 mM.
  • the weight ratio of reduced to oxidized thiol can be 10: 1 to 0.1 : 1 , more preferably 5: 1 to 1 : 1 and is most preferably about 2: 1.
  • the renaturation buffer may further contain agents to prevent self-activation of trypsinogen.
  • an inhibitor can be i.e. benzamidine, which is typically used in a concentration of 1 to 100 mM, more preferably of 5 to 10 mM.
  • the renaturation buffer may further contain agents to prevent aggregation of misfolded proteins, such as L-Arginine, which is preferably used in a concentration of 0.25 M to 1.0 M or guanidine hydrochloride in a concentration range of 0.5 M to 1.5 M.
  • Trypsinogen is typically folded in step f) by rapid dilution of the denatured protein sample with renaturation buffer.
  • the initial concentration of denatured trypsinogen after purification of the solubilized inclusion bodies is preferably from 0.5 to 50 mg/ml, even more preferably from 5 to 10 mg/ml.
  • the final concentration of trypsin after dilution is preferably from 0.01 to 1.0 mg ml, more preferably from 0.02 to 0.5 mg/ml, most preferably from 0.05 to 0.2 mg/ml.
  • the trypsinogen is folded using the pulse renaturation method as described in US 4,933,434.
  • the final concentration of trypsinogen in the renaturation buffer is typically from 0.01 to 1.0 mg/ml, preferably between 0.05 and 0.6 mg/ml and most prefer- ably between 0.2 and 0.5 mg/ml. Each added aliquot may increment the concentration of trypsinogen in the renaturation buffer by 0.01 to 1.0 mg/ml, more preferably 0.02 to 0.5 mg/ml and most preferably from 0.05 to 0.2 mg/ml.
  • the concentration of the unfolded protein can therefore be kept low, thus limiting aggregation, whereas the final volume of the reaction mixture containing the refolded trypsinogen can also be kept low.
  • the degree of trypsinogen recovery i.e. the amount of correctly folded trypsinogen resulting from renaturation in step f) compared to the amount of trypsinogen from solubilization in step d) that was used for renaturation, is at least 5 wt.%, preferably between 10 and 80 wt.%, more preferably between 15 and 50 wt.%.
  • renaturation rates of 15 wt.% and above can be achieved.
  • renaturation yields generally only 3-5 wt.% of correctly folded trypsinogen.
  • renaturation can be achieved by so called on-column folding.
  • the denatured protein is loaded onto a chromatographic column.
  • the trypsinogen protein contains an affinity tag such as a His-tag as described above
  • the denatured protein may be loaded onto an affinity matrix such as a Ni-NTA or Co-NTA.
  • the protein is refolded on the column, e.g., by applying appropriate buffer gradients as described by Epstein and Anfinsen, 1962, J. Biol. Chem., 237, 2175 and Oshima et al., 2008, J. Bioscience Bioengineering, 106, 345.
  • the trypsinogen has to be processed into trypsin (step g)).
  • the renatured trypsinogen may initially be further purified to remove precipitated, incorrectly folded trypsinogen. This can be achieved by filtration, centrifugation or chromatographic methods, preferably by filtration.
  • the protein can then be further purified by chromato- graphic methods such as size exclusion chromatography, ion exchange chromatography or affinity chromatography, more preferably cationic exchange chromatography, typically using a NaCl gradient for elution of protein. A combination of two or more purification methods may also be used.
  • removing precipitated, incorrectly folded trypsinogen and purification of trypsinogen may also be achieved in one single step, e.g. by using one of the above-mentioned chromato- graphic methods.
  • Processing of trypsinogen into trypsin is generally achieved by autocatalytic cleavage of trypsinogen into trypsin or by incubation of trypsinogen with the protease enterokinase, preferably by autocatalytic cleavage.
  • the N- terminal peptide which in the preferred embodiment of the invention consists of amino acids 13 to 23 of human trypsin, is cleaved off to result in the active trypsin product.
  • the present invention further relates to a process for obtaining a correctly folded trypsinogen, which is processed by enzymatic cleavage in order to release the active trypsin product.
  • processing of trypsinogen into trypsin may further be used for removing a tag such as an affinity tag, e.g. a His-tag.
  • a tag such as an affinity tag, e.g. a His-tag.
  • an affinity tag is located at the N-terminus of trypsinogen such that the tag is removed together with the N-terminal peptide in step g).
  • a proteolytic cleavage site may be introduced between trypsinogen and the affinity tag such that the affinity tag can be removed by proteolytic cleavage.
  • cleavage buffer should have a pH of 7 to 9, preferably of 7.5 to 8.5 and CaCl 2 in a concentration of between 10 and 50 mM.
  • the protein is then typically incubated at 20°C to 37°C until the trypsinogen is completely converted to trypsin as described by Kay and Kassell, 1971 , J. Biol. Chem., 216, 6661.
  • Suitable conditions for proteolytic digestion of trypsinogen with enterokinase are given e.g. in Grant and Hermon-Taylor, 1975, Biochem. J., 147, 363.
  • the degree of trypsin recovery i.e. the amount of correctly folded trypsin resulting from processing of trypsinogen in step g) compared to the amount of trypsinogen resulting from solubilization in step d) is at least 2.5 wt.%, preferably between 5 and 70 wt.%, more preferably between 10 and 40 wt.%, based on the amount of trypsinogen resulting from step d).
  • yields of trypsin after processing of tryp- sinogen in step g) is generally only 1-2 wt.%.
  • the activated trypsin can then optionally be further purified by using one or more of the purification methods as described above, preferably by ion exchange chromatography, affinity chromatography or size exclusion chromatography. More preferably, the affinity chromatography using ben- zamidine-sepharose is used to separate active trypsin from non-activated and inactive trypsinogen, e.g. such as described in Hanquier et al., 2003, Appl. Env. Microbiology, 69, 1 108.
  • the purified trypsin has an activity of 150-180 TAME (N Corp-p-Tosyl-L-arginine methyl ester hydrochloride) units/mg protein (Baines et al., 1964, Biochem. J., 90, 470).
  • the trypsin can then be crystallized and/or lyophilized according to standard procedures.
  • trypsin is commonly used in biological research during proteomics experiments to digest proteins into peptides for mass spectrometry analysis, e.g. in-gel digestion. Trypsin is particularly suited for this, since it has a very well defined specificity, as it hydrolyzes only the peptide bonds in which the carbonyl group is contributed either by an Arg or Lys residue.
  • Trypsin is also used to process fusion proteins.
  • fusion proteins contain a tag such as an affinity tag, e.g. a His-tag, to purify and/or identify the fusion protein and the protein part of interest.
  • Engineering a proteolytic cleavage site between the tag and the rest of the fusion protein allows trypsin to cleave and remove the tag to further characterize the protein of interest.
  • the trypsin product of the invention is used for processing of an insulin precursor such as proinsulin, preferably human proinsulin, in a process for manufacturing insulin, preferably recombinant insulin.
  • an insulin precursor such as proinsulin, preferably human proinsulin
  • insulin preferably recombinant insulin.
  • the insulin C-peptide or any other peptide located between the A- and B-chains of insulin is cleaved off by action of trypsin.
  • trypsin can also be used to dissolve blood clots in its microbial form and treat inflammation in its pancreatic form. Trypsin is further used in baby food to pre-digest it. It can break down the protein molecules, which helps the baby to digest it, as its stomach is not strong enough to digest bigger protein molecules.
  • the E. coli strain C41 (DE3) is transformed with an expression plasmid derived from pQE-T7 (Qiagen) containing the gene of SEQ ID No. 1 and coding for amino acids 13 to 247 of the human trypsin zymogen (trypsinogen) under control of the T7 promoter.
  • pQE-T7 Qiagen
  • trypsinogen trypsinogen
  • the E. coli strain C41 (DE3) is transformed with an expression plasmid derived from pQE-T7 (Qiagen) containing the gene of SEQ ID No. 1 and coding for amino acids 13 to 247 of the human trypsin zymogen (trypsinogen) under control of the T7 promoter.
  • pQE-T7 Qiagen
  • trypsinogen trypsinogen
  • coli strain is grown in a shaking flask at 37°C overnight in bioreactor medium (25 g/1 glycerol, 13.3 g/1 KH 2 P0 4 , 4 g/1 (NH 4 ) 2 S0 4 , 4.5 mg/1 thiamin HC1, 1.2 g/1 MgS0 4 7H 2 0, 8.4 mg/1 EDTA, 30 ⁇ g/ml kanamycine, 5 g/1 amino acid mix, 1 ml/1 trace elements, 2 ml/1 antifoam).
  • the amino acid mix consists of equal (per weight) amounts of the amino acids glutamic acid, glutamine, leucine, histi- dine, serine, proline, arginine, glycine and tyrosine.
  • the overnight culture ( ⁇ about 3) was used to inoculate bioreactor medium (volume ratio 1 :8.5) in a bioreactor.
  • the culture was grown to OD600 about 2 and expression was induced by ad- dition of IPTG to 1 mM final concentration.
  • the culture was further incubated until reaching an OD 600 about 30-35.
  • the fermentation broth (180 ml) is centrifuged (4,500 g / 20 min).
  • the cell pellet (about 6.4 g wet cell weight) is resuspended in 40 ml buffer (50 mM Tris HC1 pH 8.0; 200 mM NaCl; 1 mM EDTA; 1 mg/ml lysozyme) and incubated at 30°C for 1 hr on a shaker.
  • the cells are lysed by sonication (e.g. 4x30 sec with 30 sec pause on ice) followed by centrifugation (10,000 g / 20 min / 4°C).
  • the pellet is resuspended in 40 ml buffer (50 mM Tris HCl pH 8.0; 200 mM NaCl; 2.0 M urea) and centrifuged (10,000 g / 20 min / 4°C).
  • the pellet is resuspended in 40 ml buffer (50 mM Tris HC1 pH 8.0; 200 mM NaCl) and centrifuged (10,000 g / 20 min / 4°C).
  • the inclusion bodies may be stored as pellets at -80°C until further use. d Solubilization of inclusion bodies
  • the pellet of inclusion bodies (corresponding to 320 mg wet cell weight) is resuspended in 3 ml buffer (8 M urea; 50 mM glycine pH 9.0; 2 mM EDTA; 10 mM DTT). After incubation for 2 hrs at 37°C on a shaker the suspension is centrifuged (10,000 g / 20 min/ 4°C) and the supernatant isolated. e) Purification of trypsinogen
  • cationic exchange chromatography Purification of trypsinogen is performed by cationic exchange chromatography as follows. The supernatant is diluted with buffer (8 M urea; 50 mM glycine pH 9.0; 2 mM EDTA; 10 mM DTT) to a total protein concentration of 6 mg/ml and adjusted with 1 N HC1 to pH 3.0. The sample is loaded on a cationic exchange column (SP-Sepharose) pre-equilibrated in low-salt buffer (8 M urea; 50 mM glycine pH 3.0; 10 mM ⁇ -mercaptoethanol).
  • SP-Sepharose cationic exchange column
  • the protein is eluted with a linear gradient from low-salt buffer to high-salt buffer (8 M urea; 50 mM glycine pH 3.0; 10 mM ⁇ - mercaptoethanol; 1.0 M NaCl).
  • the purification using this column yields an almost 100 % recovery rate of trypsinogen und, thus, surprisingly leads only to a marginal loss of trypsinogen protein.
  • the pH of the eluted protein is adjusted to pH 9.0 by addition of 1 N NaOH before renaturation. fj Renaturation of trypsinogen
  • the protein is renatured by dilution (final protein concentration about 0.05 mg/ml) into folding buffer (20 mM Tris HCl pH 8.25; 2.0 mM ⁇ -mercaptoethanol; 1.0 mM 2-hydroxyethyldisulfide; 10 mM benzamidine; 1.0 M L-arginine) followed by incubation for 1 hr at 4°C and subsequent dialy- sis against 20 mM Tris HCl pH 8.0 containing 10 mM benzamidine at 4°C. The sample is finally dialyzed against 20 mM Na citrate pH 3.0 containing 5 mM benzamidine and subsequently centri- fuged (10,000 g/ 10 min/4°C) to remove precipitated material. NaCl is added to the protein solution to a concentration of 0.375 M and the protein sample is filtered through a 0.22 ⁇ filter.
  • the protein is then further purified by cationic exchange chromatography (SP-sepharose) by applying the protein sample to a column pre-equilibrated in a 20 mM Na citrate pH 3.0 and eluted with a gradient to the buffer 20 mM Na citrate pH 3.0 containing 1.5 M NaCl.
  • SP-sepharose cationic exchange chromatography
  • Active trypsin is then purified from non-active trypsin or residual amounts of trypsinogen by affinity chromatography using a benzamidine-sepharose column.
  • the autoactivation reaction is directly loaded on the column pre-equilibrated with buffer (100 mM Tris HC1 pH 8.0; 10 mM CaCl 2 ). Contaminants are removed by washing the column in washing buffer (100 mM Tris HCl pH 8.0; 10 mM CaCl 2 ; 1.0 M NaCl).
  • the protein is eluted with 20 mM Na citrate pH 3.0.
  • Example 2 This example differs from the process described in Example 1 by using size exclusion chromatography for purification of trypsinogen in step e).
  • the supernatant is diluted with buffer (8 M urea; 50 mM glycine pH 9.0; 2 mM EDTA; 10 mM DTT) to a total protein concentration of 10 mg/ml and filtered through a 0.22 ⁇ filter.
  • the sam- pie is loaded on a gel filtration column (Sephacryl SI 00) pre-equilibrated in buffer (8 M urea; 50 mM glycine pH 9.0; 10 mM ⁇ -mercaptoethanol).
  • the protein is eluted with the same buffer and it can be directly used for renaturation.
  • This example differs from the process described in Example 1 by using size anionic exchange chromatography for purification of trypsinogen in step e).
  • the supernatant is diluted with buffer (8 M urea; 50 mM glycine pH 9.0; 2 mM EDTA; 10 mM DTT) to a total protein concentration of 6 mg/ml.
  • the sample is loaded on an anionic exchange column (Q-Sepharose) pre-equilibrated in (8 M urea; 50 mM glycine pH 9.0; 10 mM ⁇ -mercaptoethanol).
  • the protein is eluted with a linear gradient from low-salt buffer to high-salt buffer (8 M urea; 50 mM glycine pH 9.0; 10 mM ⁇ -mercaptoethanol; 1.0 M NaCl).
  • the eluted protein can be used directly for renaturation.
  • This example differs from the process described in Example 1 by using metal affinity chromato- graphy (MAC) for purification of trypsinogen in step e).
  • MAC metal affinity chromato- graphy
  • the expressed trypsinogen contains an N-terminal polyhistidine-tag
  • the supernatant is further diluted at least 10-fold with buffer (8 M urea; 50 mM glycine pH 9.0; 5 mM ⁇ -mercaptoethanol; 10 mM imidaz- ole) and filtered through a 0.22 ⁇ filter.
  • the protein is loaded onto a Ni-NTA column pre- equilibrated in dilution buffer.
  • the protein is then eluted from the column with a gradient to a buffer (8 M urea; 50 mM glycine pH 9.0; 5 mM ⁇ -mercaptoethanol) containing 1 M imidazole.
  • the eluted protein can be directly used for renaturation.
  • This example differs from the process described in Example 1 by using pulse renaturation in step f).
  • the protein is added stepwise to the renaturation buffer, beginning with an amount of protein corresponding to a final concentration of 0.05 mg/ml in the renaturation buffer. After 1 hr incubation at 4°C, the same amount of protein is added to the renaturation buffer resulting in a total protein concentration of 0.10 mg/ml. After 1 hr incubation at 4°C, more protein is added and the process is repeated until the total protein concentration is 0.25 mg ml.
  • Example 5 This example is included to illustrate the significance of using purified trypsinogen for renaturation.
  • the example differs from Example 5 in that the solubilized protein of step d) is directly used for pulse renaturation of trypsinogen without any further purification.
  • the following section il- lustrates the benefit of purification of trypsinogen prior to renaturation (all percentages are in weight percent):
  • the purity of trypsinogen is defined here as that part of the OD ⁇ measurement that can be attributed to trypsinogen assuming an extinction coefficient of -1.5 ml mg 'cm *1 .
  • the purity of trypsinogen and trypsin is judged based on SDS gel electrophoresis.
  • the recovery at this processing step is estimated by the ratio of amount of protein determined by Bradford before and after the processing step.
  • the recovery rate at this processing step is calculated with respect to the amount of trypsinogen in the purified and non-purified protein sample.
  • the yield of trypsinogen after renaturation and the yield of trypsin after processing was increased by 500 wt.%.
  • the efficiency of renaturation was enhanced resulting in an overall higher yield of trypsinogen, and, after processing, also in a higher yield of active trypsin product.

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Abstract

La présente invention concerne un procédé de production de trypsine recombinante et son utilisation en vue de la transformation d'un précurseur de l'insuline en insuline. L'invention concerne, en particulier, un procédé de production de trypsine recombinante à partir de trypsinogène recombinant produit dans une cellule hôte procaryote. Dans le procédé de l'invention, le trypsinogène recombinant est produit par la cellule hôte sous la forme de corps d'inclusion. Le trypsinogène contenu dans les corps d'inclusion est purifié, puis replié pour retrouver sa conformation native avec formation de ponts disulfure. Le trypsinogène replié subit ensuite une nouvelle transformation pour donner de la trypsine active.
PCT/EP2012/000497 2011-02-04 2012-02-03 Procédé de production de trypsine recombinante WO2012104099A1 (fr)

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CN104694522A (zh) * 2015-02-16 2015-06-10 中国人民解放军军事医学科学院放射与辐射医学研究所 一种重组乙酰化阳离子型胰蛋白酶的制备方法及其应用
WO2015150799A1 (fr) * 2014-03-31 2015-10-08 Enzymatica Ab Nouvelles méthodes, polypeptides et utilisations associées
WO2016081289A1 (fr) 2014-11-18 2016-05-26 Merck Sharp & Dohme Corp. Procédé de préparation de trypsine recombinante
CN106232810A (zh) * 2014-01-29 2016-12-14 安吉酶迪卡公司 新的治疗
CN113897292A (zh) * 2020-06-22 2022-01-07 新疆维吾尔自治区疾病预防控制中心 一种融合蛋白纯化的制备方法
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102994600A (zh) * 2012-12-11 2013-03-27 鲁南新时代生物技术有限公司 甘精胰岛素前体的酶切转化方法
CN106232810A (zh) * 2014-01-29 2016-12-14 安吉酶迪卡公司 新的治疗
WO2015150799A1 (fr) * 2014-03-31 2015-10-08 Enzymatica Ab Nouvelles méthodes, polypeptides et utilisations associées
CN106257988A (zh) * 2014-03-31 2016-12-28 安吉酶迪卡公司 新颖方法、多肽以及其用途
JP2017511133A (ja) * 2014-03-31 2017-04-20 エンツィマティカ アクティエ ボラーグ 新規の方法、ポリペプチド、及びその使用
US20170107503A1 (en) * 2014-03-31 2017-04-20 Enzymatica Ab Novel methods, polypeptides and uses thereof
WO2016081289A1 (fr) 2014-11-18 2016-05-26 Merck Sharp & Dohme Corp. Procédé de préparation de trypsine recombinante
EP3221448A4 (fr) * 2014-11-18 2018-05-09 Merck Sharp & Dohme Corp. Procédé de préparation de trypsine recombinante
US10947521B2 (en) 2014-11-18 2021-03-16 Merck Sharp & Dohme Corp. Process for producing recombinant trypsin
CN104694522A (zh) * 2015-02-16 2015-06-10 中国人民解放军军事医学科学院放射与辐射医学研究所 一种重组乙酰化阳离子型胰蛋白酶的制备方法及其应用
CN113897292A (zh) * 2020-06-22 2022-01-07 新疆维吾尔自治区疾病预防控制中心 一种融合蛋白纯化的制备方法
WO2022069903A1 (fr) * 2020-10-01 2022-04-07 Ipsen Biopharm Limited Procédé de production de bêta-trypsine

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