WO2023030562A1 - Genetically modified bacterial strain e. coli producing clostripain peptidase - Google Patents

Genetically modified bacterial strain e. coli producing clostripain peptidase Download PDF

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WO2023030562A1
WO2023030562A1 PCT/CZ2022/050081 CZ2022050081W WO2023030562A1 WO 2023030562 A1 WO2023030562 A1 WO 2023030562A1 CZ 2022050081 W CZ2022050081 W CZ 2022050081W WO 2023030562 A1 WO2023030562 A1 WO 2023030562A1
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clostripain
collagenase
isolation
peptidase
ccm
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English (en)
French (fr)
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Martin BENESIK
Marek MOSA
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Mb Pharma SRO
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Mb Pharma SRO
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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
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • 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
    • 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
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/22—Cysteine endopeptidases (3.4.22)
    • C12Y304/22008—Clostripain (3.4.22.8)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/145—Clostridium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • ColG and ColH need to act together in a specific ratio and require the synergic effect of additional enzymes as neutral proteases or thermolysin (O'Gorman et al., 2005; G. HJ. Wolters et al., 1992).
  • additional enzymes as neutral proteases or thermolysin
  • ColH and ColG can cleave the pancreatic tissue individually or alone with an admixture of neutral proteases, there are studies on the efficient ratios of these two forms of C. histolyticum collagenases.
  • the ideal ratio of ColH and ColG is said to be 0.64:1.0 (Brandhorst et al., 2008), which indicates that in this combination, ColG is essential for the cleaving.
  • This gene was cloned to the expression plasmid with the use of restriction cloning (Ncol and BamHI) and a histidine anchor was placed at the C-terminus of the protein sequence.
  • the tag removal site was not integrated since the protein is functional without the removal as well.
  • the theoretical isoelectric point of the protein including the his-tag is 8.31 and the molecular weight (mw) is 45.7 kDa.
  • the amino acid sequence analysis indicates that the protein comprises a cystein-peptidase domain belonging to the peptidase family Cll ( Figure 4), which is referred to as the clostripain family of enzymes and contains chymotripsin-like specific sequences (Barrett & Rawlings, 2014).
  • Protein repurification A protein with a histidine anchor is purified by means of metallochelation chromatography. A HisTrap HP 5ml (filled and charged with Ni Sepharose High Performance) is used for this purpose. The methodology of column equilibration, sample application and sample elution is the same for all the systems. The used buffers and water must be deaerated before the purification for 10-15 minutes in an ultrasonic bath. The abbreviation CV, used below, refers to the column volume. The column is equilibrated with at least 5 CV of the buffer to adjust suitable conditions for the purification (50 mM Tris, 300 mM NaCI, 10 mM imidazole, pH 7.5).
  • Figure 1 shows the amino acid sequence of the clostripain-like protein according to the invention, produced by the strain E. coli CCM 9176.
  • the genetically modified strain maintains the sequence established by sequencing of the strain Clostridium histolyticum CCM 8656, described in the Czech invention application PV 2017-537, for which the patent CZ 308157 was granted.
  • the isolated islets were cultivated in the CMRL-1066 media supplemented with 10% FBS, 5% HEPES, 1% Glutamax and 1% penicillin-streptomycin in a moisturizing incubator at 37 °C and 5% CO2 atmosphere.
  • the quality of the isolated IL was determined with the use of vital dyeing and stimulated secretion of insulin.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Cell Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
PCT/CZ2022/050081 2021-08-30 2022-08-23 Genetically modified bacterial strain e. coli producing clostripain peptidase Ceased WO2023030562A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CZ2021-400 2021-08-30
CZ2021-400A CZ2021400A3 (cs) 2021-08-30 2021-08-30 Kmen produkující clostripain-like protein pro použití ve směsi s rekombinantní kolagenázou ColG a ColH pro izolaci Langerhansových ostrůvků pankreatu

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WO2023030562A1 true WO2023030562A1 (en) 2023-03-09

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ2017537A3 (cs) 2017-09-13 2018-10-10 MB PHARMA s.r.o. Kmen bakterie Clostridium histolitycum, kolagenáza připravená s použitím tohoto kmene a její použití

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2193354A1 (en) * 1995-04-25 1996-10-31 Catherine Lee Composition containing collagenase and chymopapain for isolating hepatocytes and pancreatic islet cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CZ2017537A3 (cs) 2017-09-13 2018-10-10 MB PHARMA s.r.o. Kmen bakterie Clostridium histolitycum, kolagenáza připravená s použitím tohoto kmene a její použití
WO2019052586A1 (en) * 2017-09-13 2019-03-21 MB PHARMA s.r.o. BACTERIAL STRAIN CLOSTRIDIUM HISTOLYTICUM AND USE THEREOF
CZ308157B6 (cs) 2017-09-13 2020-01-29 MB PHARMA s.r.o. Kmen bakterie Clostridium histolyticum, kolagenáza připravená s použitím tohoto kmene a její použití

Non-Patent Citations (25)

* Cited by examiner, † Cited by third party
Title
ALIPOUR, H.RAZ, A.DINPARAST DJADID, N.RAMI, A.MAHDIAN, S. M. A.: "Codon optimization of Col H gene encoding Clostridium histolyticum collagenase to express in Escherichia coli", CODON OPTIMIZATION OF COL H GENE ENCODING CLOSTRIDIUM HISTOLYTICUM COLLAGENASE TO EXPRESS IN ESCHERICHIA COLI, 2014, Retrieved from the Internet <URL:https://doi.org/10.7287/peerj.preprints.754>
BARRETT, A. J.RAWLINGS, N. D.: "Evolutionary Lines of Cysteine Peptidases", BIOLOGICAL, 2014, Retrieved from the Internet <URL:https://doi.Org/10.1515/bchm.2001.382.5.727>
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BRANDHORST, H., BRANDHORST, D., HESSE, F., AMBROSIUS, D., BRENDEL, M., KAWAKAMI, Y., BRETZEL, R. G.: "Successful human islet isolation utilizing recombinant collagenase", DIABETES, 2003, Retrieved from the Internet <URL:https://doi.Org/10.2337/diabetes.52.5.1143>
BRANDHORST, H., FRIBERG, A., ANDERSSON, H. H., FELLDIN, M., FOSS, A., SALMELA, K., LUNDGREN, T., TIBELL, A., TUFVESON, G., KORSGRE: " The importance of tryptic-like activity in purified enzyme blends for efficient islet isolation", TRANSPLANTATION, 2009, Retrieved from the Internet <URL:https://doi.org/10.1097/TP.0b013e31819499f0>
BRANDHORST, H., JOHNSON, P. R., MONCH, J., KURFURST, M., KORSGREN, O., BRANDHORST, D.: "Comparison of Clostripain and Neutral Protease as Supplementary Enzymes for Human Islet Isolation", CELL TRANSPLANTATION, 2019, Retrieved from the Internet <URL:https://doi.org/10.1177/0963689718811614>
BRANDHORST, HRAEMSCH-GUENTHER, N.RAEMSCH, C.FRIEDRICH, O.HUETTLER, S.KURFUERST, M.KORSGREN, O.BRANDHORST, D.: "The ratio between collagenase class I and class II influences the efficient islet release from the rat pancreas", TRANSPLANTATION, 2008, Retrieved from the Internet <URL:https://doi.org/10.1097/TP.0b013e31816050c8>
DATABASE UNIPROT [online] 31 July 2019 (2019-07-31), ANONYMOUS: "Alpha-clostripain-like protein", XP093004070, Database accession no. A0A4U9R4H2 *
DENDO M. ET AL.: "SDC, Materials and Methods", 1 July 2015 (2015-07-01), XP002808121, Retrieved from the Internet <URL:https://cdn-links.lww.com/permalink/tp/b/tp_2014_12_17_dendo_tpa-2014-0332_sdc1.pdf> [retrieved on 20221130] *
DENDO, M., MAEDA, H., YAMAGATA, Y., MURAYAMA, K., WATANABE, K., IMURA, T., INAGAKI, A., IGARASHI, Y., KATOH, Y., EBINA, M., FUJIMO: "Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation", TRANSPLANTATION, 2015, Retrieved from the Internet <URL:https://doi.org/10.1097/TP.0000000000000662>
DUCKA, P., ECKHARD, U., SCHONAUER, E., KOFLER, S., GOTTSCHALK, G., BRANDSTETTER, H., NUSS, D.: " A universal strategy for high-yield production of soluble and functional clostridial collagenases in E. coli.", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 2009, Retrieved from the Internet <URL:https://doi.org/10.1007/s00253-009-1953-4>
JUNG, C. M., MATSUSHITA, O., KATAYAMA, S., MINAMI, J., OHHIRA, I., & OKABE, A.: "Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification", MICROBIOLOGY AND IMMUNOLOGY, 1996, Retrieved from the Internet <URL:https://doi.org/l0.l111/j.1348-0421.1996.tb01161.x>
KIM ET AL: "Secretory expression of active clostripain in Escherichia coli", JOURNAL OF BIOTECHNOLOGY, ELSEVIER, AMSTERDAM NL, vol. 131, no. 3, 6 September 2007 (2007-09-06), pages 346 - 352, XP022232039, ISSN: 0168-1656, DOI: 10.1016/J.JBIOTEC.2007.07.936 *
KIM, C. K.LEE, S. Y.KWON, O. J.LEE, S. M.NAH, S. Y.JEONG, S. M: "Secretory expression of active clostripain in Escherichia coli", JOURNAL OF BIOTECHNOLOGY, 2007, Retrieved from the Internet <URL:https://doi.Org/10.1016/j.jbiotec.2007.07.936>
KLOCK, G., KOWALSKI, M. B., HERING, B. J., EIDEN, M. E., WEIDEMANN, A., LANGER, S., ZIMMERMANN, U., FEDERLIN, K., BRETZEL, R. G.: "Fractions from commercial collagenase preparations: Use in enzymic isolation of the islets of Langerhans from porcine pancreas", CELL TRANSPLANTATION, 1996, Retrieved from the Internet <URL:https://doi.org/10.1016/0963-6897(96)00023-1>
LOGANATHAN, G., SUBHASHREE, V., BREITE, A. G., TUCKER, W. W., NARAYANAN, S., DHANASEKARAN, M., MOKSHAGUNDAM, S. P., GREEN, M. L., : "Beneficial effect of recombinant rClrC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield", AMERICAN JOURNAL OF TRANSPLANTATION, 2018, Retrieved from the Internet <URL:https://doi.org/l0.1111/ajt.14542>
MAMI DENDO ET AL: "Synergistic Effect of Neutral Protease and Clostripain on Rat Pancreatic Islet Isolation :", TRANSPLANTATION, vol. 99, no. 7, 1 July 2015 (2015-07-01), GB, pages 1349 - 1355, XP055524870, ISSN: 0041-1337, DOI: 10.1097/TP.0000000000000662 *
O'GORMAN, DKIN, T.MCGHEE-WILSON, D.SHAPIRO, A. M. J.LAKEY, J. R. T.: "Multi-lot analysis of custom collagenase enzyme blend in human islet isolations", TRANSPLANTATION PROCEEDINGS, 2005, Retrieved from the Internet <URL:https://doi.org/10.1016/j.transproceed.2005.09.139>
STAHLE, M.FOSS, A.GUSTAFSSON, B.LEMPINEN, M.LUNDGREN, T.RAFAEL, ETUFVESON, G.KORSGREN, O.FRIBERG, A.: "Clostripain, the Missing Link in the Enzyme Blend for Efficient Human Islet Isolation", TRANSPLANTATION DIRECT, 2015, Retrieved from the Internet <URL:https://doi.org/10.1097/txd.0000000000000528>
VARGAS, F., VIVES-PI, M., SOMOZA, N., ARMENGOL, P., ALCALDE, L., MARTI, M., COSTA, M., SERRADELL, L., DOMINGUEZ, O., FERNANDEZ-LLA: "Endotoxin contamination may be responsible for the unexplained failure of human pancreatic islet transplantation", TRANSPLANTATION, 1998, Retrieved from the Internet <URL:https://doi.org/10.1097/00007890-199803150-00020>
VOLPE, L., SALAMONE, M., GIARDINA, A., GHERSI, G.: "Optimization of a Biotechnological Process for Production and Purification of Two Recombinant Proteins: Col G and Col H", CHEMICAL ENGINEERING TRANSACTIONS, 2016, Retrieved from the Internet <URL:https://doi.org/10.3303/CET1649011>
VOS-SCHEPERKEUTER, G. H.VAN SUYLICHEM, P. T. RVONK, M. W. A.WOLTERS, G. H. JVAN SCHILFGAARDE, R.: "Histochemical Analysis of the Role of Class I and Class II Clostridium Histolyticum Collagenase in the Degradation of Rat Pancreatic Extracellular Matrix for Islet Isolation", CELL TRANSPLANTATION, 1997, Retrieved from the Internet <URL:https://doi.org/10.1177/096368979700600407>
WITTE, V., WOLF, N., DIEFENTHAL, T., REIPEN, G., DARGATZ, H.: "Heterologous expression of the clostripain gene from Clostridium histolyticum in Escherichia coli and Bacillus subtilis: Maturation of the clostripain precursor is coupled with self-activation", MICROBIOLOGY, 1994, Retrieved from the Internet <URL:https://doi.org/10.1099/13500872-140-5-1175>
WOLTERS, G. H.J., VOS -SCHEPERKEUTER, G. H., VAN DEIJNEN, J. H. M., VAN SCHILFGAARDE, R.: " An analysis of the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet isolation", DIABETOLOGIA, 1992, Retrieved from the Internet <URL:https://doi.org/10.1007/BF00429093>
WOLTERS, GERRIT H.J., VOS-SCHEPERKEUTER, G. H., LIN, H. C., & VAN SCHILFGAARDE, R.: "Different roles of class I and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation", DIABETES, 1995, Retrieved from the Internet <URL:https://doi.org/10.2337/diab.44.2.227>

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CZ2021400A3 (cs) 2023-03-29

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