WO2003040335A3 - Engineering of leader peptides for the secretion of recombinant proteins in bacteria - Google Patents

Engineering of leader peptides for the secretion of recombinant proteins in bacteria Download PDF

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Publication number
WO2003040335A3
WO2003040335A3 PCT/US2002/035618 US0235618W WO03040335A3 WO 2003040335 A3 WO2003040335 A3 WO 2003040335A3 US 0235618 W US0235618 W US 0235618W WO 03040335 A3 WO03040335 A3 WO 03040335A3
Authority
WO
WIPO (PCT)
Prior art keywords
leader peptides
export
proteins
folded
cytoplasm
Prior art date
Application number
PCT/US2002/035618
Other languages
French (fr)
Other versions
WO2003040335A2 (en
Inventor
George Georgiou
Matthew Delisa
Original Assignee
Res Dev Foundation
George Georgiou
Matthew Delisa
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
Application filed by Res Dev Foundation, George Georgiou, Matthew Delisa filed Critical Res Dev Foundation
Priority to AU2002360348A priority Critical patent/AU2002360348B8/en
Priority to EP02795597A priority patent/EP1451367A4/en
Priority to CA002465724A priority patent/CA2465724A1/en
Priority to JP2003542582A priority patent/JP2005522188A/en
Publication of WO2003040335A2 publication Critical patent/WO2003040335A2/en
Publication of WO2003040335A3 publication Critical patent/WO2003040335A3/en

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Classifications

    • 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
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1051—Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/034—Fusion polypeptide containing a localisation/targetting motif containing a motif for targeting to the periplasmic space of Gram negative bacteria as a soluble protein, i.e. signal sequence should be cleaved
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention provides methods of isolating of leader peptides capable of directing export of heterologous proteins from the bacterial cytoplasm. The methods rely on the screening of libraries of putative leader peptides or of leader peptide mutants for sequences that allow rapid export and thus can rescue a short-lived reporter protein from degradation in the cytoplasm. The mutant leader peptides identified herein are shown to confer significantly higher steady state levels of export not only for short-lived reporter protein but also for other stable, long-lived proteins. These leader peptides can be used to direct or enhance protein secretion. The present invention further discloses methods for the export of cytoplasmically folded protein via the Tat pathway. Proteins having disulfide bonds are first folded within the cytoplasm in suitable oxidizing mutant strains. Such cytoplasmically pre-folded proteins containing disulfide bonds are then exported via the Tat pathway.
PCT/US2002/035618 2001-11-05 2002-11-05 Engineering of leader peptides for the secretion of recombinant proteins in bacteria WO2003040335A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU2002360348A AU2002360348B8 (en) 2001-11-05 2002-11-05 Engineering of leader peptides for the secretion of recombinant proteins in bacteria
EP02795597A EP1451367A4 (en) 2001-11-05 2002-11-05 Engineering of leader peptides for the secretion of recombinant proteins in bacteria
CA002465724A CA2465724A1 (en) 2001-11-05 2002-11-05 Engineering of leader peptides for the secretion of recombinant proteins in bacteria
JP2003542582A JP2005522188A (en) 2001-11-05 2002-11-05 Leader peptide for promoting secretion of genetically engineered protein in bacteria and isolation method thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US60/ 2001-01-02
US33745201P 2001-11-05 2001-11-05
US60/337,452 2001-11-05

Publications (2)

Publication Number Publication Date
WO2003040335A2 WO2003040335A2 (en) 2003-05-15
WO2003040335A3 true WO2003040335A3 (en) 2003-12-31

Family

ID=23320598

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2002/035618 WO2003040335A2 (en) 2001-11-05 2002-11-05 Engineering of leader peptides for the secretion of recombinant proteins in bacteria

Country Status (3)

Country Link
EP (1) EP1451367A4 (en)
CN (1) CN100564540C (en)
WO (1) WO2003040335A2 (en)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2793424A1 (en) * 2010-03-18 2011-09-22 Cornell University Engineering correctly folded antibodies using inner membrane display of twin-arginine translocation intermediates
WO2012137187A1 (en) * 2011-04-08 2012-10-11 Anthem Biosciences Pvt Ltd Novel expression and secretion vector systems for heterologous protein production in escherichia coli
KR101470595B1 (en) 2012-08-01 2014-12-10 대구가톨릭대학교산학협력단 Extracellular Secreted GFP Gene and Vector Expressing the Gene
CN102851270A (en) * 2012-08-03 2013-01-02 江南大学 Hybrid streptomycete trypsin zymogen and application thereof
GB201713732D0 (en) * 2017-08-25 2017-10-11 Alta Innovations Ltd Tat expression system
CN109575116B (en) * 2018-11-09 2022-04-22 广东海洋大学 Mitochondrial localization leader peptide and discovery method and application thereof
CN110616227A (en) * 2019-09-30 2019-12-27 天津科技大学 Gene, recombinant expression vector, engineering strain and application of anti-freeze protein from tenebrio molitor

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110860A1 (en) * 2000-09-18 2002-08-15 Sierd Bron Twin-arginine translocation in Bacillus
US20020182672A1 (en) * 2000-10-10 2002-12-05 Marc Kolkman Enhanced secretion of a polypeptide by a microorganism

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU3243199A (en) * 1998-03-05 1999-09-20 University Of British Columbia, The Methods for assaying type iii secretion inhibitors
CA2324974A1 (en) * 1998-04-01 1999-10-14 The Governors Of The University Of Alberta Compositions and methods for protein secretion

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020110860A1 (en) * 2000-09-18 2002-08-15 Sierd Bron Twin-arginine translocation in Bacillus
US20020182672A1 (en) * 2000-10-10 2002-12-05 Marc Kolkman Enhanced secretion of a polypeptide by a microorganism

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DELISA M.P. ET AL.: "Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway", PROC. NATL. ACAD. SCI. USA, vol. 100, no. 10, 13 May 2003 (2003-05-13), pages 6115 - 6120, XP002970386 *
DELISA M.P.: "Genetic analysis of the twin arginine translocator secretion pathway in bacteria", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 33, 16 August 2002 (2002-08-16), pages 29825 - 29831, XP002970385 *
See also references of EP1451367A4 *

Also Published As

Publication number Publication date
CN1788092A (en) 2006-06-14
WO2003040335A2 (en) 2003-05-15
EP1451367A4 (en) 2006-06-14
CN100564540C (en) 2009-12-02
EP1451367A2 (en) 2004-09-01

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