WO2021188379A3 - Production of soluble recombinant protein - Google Patents

Production of soluble recombinant protein Download PDF

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Publication number
WO2021188379A3
WO2021188379A3 PCT/US2021/022126 US2021022126W WO2021188379A3 WO 2021188379 A3 WO2021188379 A3 WO 2021188379A3 US 2021022126 W US2021022126 W US 2021022126W WO 2021188379 A3 WO2021188379 A3 WO 2021188379A3
Authority
WO
WIPO (PCT)
Prior art keywords
products
protein
cytoplasm
proteins
expression
Prior art date
Application number
PCT/US2021/022126
Other languages
French (fr)
Other versions
WO2021188379A2 (en
Inventor
Min-ju CHANG
Natalia OGANESYAN
Andrew Lees
Original Assignee
Fina Biosolutions, Llc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US16/819,775 external-priority patent/US11060123B2/en
Application filed by Fina Biosolutions, Llc filed Critical Fina Biosolutions, Llc
Priority to KR1020227035931A priority Critical patent/KR20220154221A/en
Priority to JP2022555779A priority patent/JP7449000B2/en
Priority to EP21772422.8A priority patent/EP4121541A2/en
Priority to CN202180021748.8A priority patent/CN115867661A/en
Priority to US17/797,851 priority patent/US20230242961A1/en
Priority to CA3168571A priority patent/CA3168571A1/en
Priority to AU2021239914A priority patent/AU2021239914A1/en
Publication of WO2021188379A2 publication Critical patent/WO2021188379A2/en
Publication of WO2021188379A3 publication Critical patent/WO2021188379A3/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5428—IL-10
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0051—Oxidoreductases (1.) acting on a sulfur group of donors (1.8)
    • 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)
    • C12N9/485—Exopeptidases (3.4.11-3.4.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01007—Glutathione-disulfide reductase (1.8.1.7), i.e. glutathione reductase (NADPH)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y108/00—Oxidoreductases acting on sulfur groups as donors (1.8)
    • C12Y108/01—Oxidoreductases acting on sulfur groups as donors (1.8) with NAD+ or NADP+ as acceptor (1.8.1)
    • C12Y108/01008—Protein-disulfide reductase (1.8.1.8), i.e. thioredoxin
    • 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)
    • C12Y304/11—Aminopeptidases (3.4.11)
    • C12Y304/11018—Methionyl aminopeptidase (3.4.11.18)
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Virology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention is directed to methods and compositions for the expression and purification of products such as peptides and proteins in microorganisms. In particular, pre-products are expressed recombinantly, wherein the cytoplasm of the microorganism alters the expressed pre-products to produce products in an active/final or otherwise desirable form. Alterations associated with expression of a desired recombinant product include shifting of the redox state of the cytoplasm to allow proper protein folding, site-directed cleavage of pre-proteins to activate the protein, site-directed cleavage of an unwanted methionine from the N terminus of the protein, and/or one or more ligations to form desired protein configurations, all within the same cell.
PCT/US2021/022126 2019-09-13 2021-03-12 Production of soluble recombinant protein WO2021188379A2 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
KR1020227035931A KR20220154221A (en) 2020-03-16 2021-03-12 Production of soluble recombinant proteins
JP2022555779A JP7449000B2 (en) 2020-03-16 2021-03-12 Production of soluble recombinant proteins
EP21772422.8A EP4121541A2 (en) 2020-03-16 2021-03-12 Production of soluble recombinant protein
CN202180021748.8A CN115867661A (en) 2020-03-16 2021-03-12 Production of soluble recombinant proteins
US17/797,851 US20230242961A1 (en) 2019-09-13 2021-03-12 Production of Soluble Recombinant Protein
CA3168571A CA3168571A1 (en) 2020-03-16 2021-03-12 Production of soluble recombinant protein
AU2021239914A AU2021239914A1 (en) 2020-03-16 2021-03-12 Production of soluble recombinant protein

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US202062990083P 2020-03-16 2020-03-16
US16/819,775 US11060123B2 (en) 2014-01-31 2020-03-16 Production of soluble recombinant protein without n-terminal methionine
US62/990,083 2020-03-16
US16/819,775 2020-03-16
US202163152954P 2021-02-24 2021-02-24
US63/152,954 2021-02-24

Publications (2)

Publication Number Publication Date
WO2021188379A2 WO2021188379A2 (en) 2021-09-23
WO2021188379A3 true WO2021188379A3 (en) 2021-10-28

Family

ID=77772149

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/022126 WO2021188379A2 (en) 2019-09-13 2021-03-12 Production of soluble recombinant protein

Country Status (7)

Country Link
EP (1) EP4121541A2 (en)
JP (1) JP7449000B2 (en)
KR (1) KR20220154221A (en)
CN (1) CN115867661A (en)
AU (1) AU2021239914A1 (en)
CA (1) CA3168571A1 (en)
WO (1) WO2021188379A2 (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117603323A (en) * 2023-05-16 2024-02-27 张文康 Preparation method of botulinum toxin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090253900A1 (en) * 2003-12-19 2009-10-08 Novo Nordisk A/S Processing of Peptides and Proteins
US20160333057A1 (en) * 2014-01-31 2016-11-17 Fina Biosolutions, Llc Expression and Purification of CRM197 and Related Proteins

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6071718A (en) 1996-11-27 2000-06-06 Abbott Laboratories Methods of producing a recombinant protein
ATE506433T1 (en) 2003-12-19 2011-05-15 Novo Nordisk As PROCESSING OF PEPTIDES AND PROTEINS
TW201343911A (en) 2012-04-18 2013-11-01 Providence University Escherichia coli expression system for producing mature human tyrosinase and producing method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090253900A1 (en) * 2003-12-19 2009-10-08 Novo Nordisk A/S Processing of Peptides and Proteins
US20160333057A1 (en) * 2014-01-31 2016-11-17 Fina Biosolutions, Llc Expression and Purification of CRM197 and Related Proteins

Also Published As

Publication number Publication date
KR20220154221A (en) 2022-11-21
CN115867661A (en) 2023-03-28
JP7449000B2 (en) 2024-03-13
CA3168571A1 (en) 2021-09-23
AU2021239914A1 (en) 2022-09-08
EP4121541A2 (en) 2023-01-25
WO2021188379A2 (en) 2021-09-23
JP2023517708A (en) 2023-04-26

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