WO2000018881A3 - Intein mediated peptide ligation - Google Patents

Intein mediated peptide ligation Download PDF

Info

Publication number
WO2000018881A3
WO2000018881A3 PCT/US1999/022776 US9922776W WO0018881A3 WO 2000018881 A3 WO2000018881 A3 WO 2000018881A3 US 9922776 W US9922776 W US 9922776W WO 0018881 A3 WO0018881 A3 WO 0018881A3
Authority
WO
WIPO (PCT)
Prior art keywords
protein
intein
target protein
synthetic
peptide
Prior art date
Application number
PCT/US1999/022776
Other languages
French (fr)
Other versions
WO2000018881A2 (en
WO2000018881A8 (en
WO2000018881A9 (en
Inventor
Ming-Qun Xu
Thomas C Evans
Original Assignee
New England Biolabs Inc
Xu Ming Qun
Thomas C Evans
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 New England Biolabs Inc, Xu Ming Qun, Thomas C Evans filed Critical New England Biolabs Inc
Priority to DE69931382T priority Critical patent/DE69931382T2/en
Priority to CA2344764A priority patent/CA2344764C/en
Priority to JP2000572329A priority patent/JP4749548B2/en
Priority to EP99950063A priority patent/EP1117693B1/en
Priority to US09/786,009 priority patent/US7001745B1/en
Publication of WO2000018881A2 publication Critical patent/WO2000018881A2/en
Publication of WO2000018881A3 publication Critical patent/WO2000018881A3/en
Publication of WO2000018881A8 publication Critical patent/WO2000018881A8/en
Publication of WO2000018881A9 publication Critical patent/WO2000018881A9/en

Links

Classifications

    • 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
    • C12N9/93—Ligases (6)
    • 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
    • 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
    • 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
    • C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

An in vitro method for producing a semi-synthetic fusion protein is provided, whereby a target protein fused to an intein - a protein splicing element - is selectively cleaved in a first step as depicted in Figure 1 with a thiol reagent, forming a carboxyl-terminal thioester of the target protein and releasing the target protein from the intein. In a subsequent step as shown in Figure 1, a desired, synthetic, protein or peptide having an amino-terminal cysteine is ligated to the target protein. Standard thiol-reagents such as DTT, or thiol-reagents optimized for ligation such as the odorless MESNA, may be used in the first step. The method permits the direct ligation of a desired peptide to a thioester bond that had linked a target protein to an intein. An in vivo variation of the method will permit production of a cytotoxic protein: a truncated, inactive, form of the protein fused to an intein is introduced in vivo, this fusion product is then selectively cleaved, and a synthetic protein or peptide is subsequently ligated at a carboxyl-terminal thioester of the target protein in order to restore the native activity of the cytotoxic protein.
PCT/US1999/022776 1998-09-30 1999-09-30 Intein mediated peptide ligation WO2000018881A2 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
DE69931382T DE69931382T2 (en) 1998-09-30 1999-09-30 INTEGRATED PEPTIDE LIGATION
CA2344764A CA2344764C (en) 1998-09-30 1999-09-30 Intein mediated peptide ligation
JP2000572329A JP4749548B2 (en) 1998-09-30 1999-09-30 Intein-mediated peptide linkage
EP99950063A EP1117693B1 (en) 1998-09-30 1999-09-30 Intein mediated peptide ligation
US09/786,009 US7001745B1 (en) 1998-09-30 1999-09-30 Intein mediated peptide ligation

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10241398P 1998-09-30 1998-09-30
US60/102,413 1998-09-30

Publications (4)

Publication Number Publication Date
WO2000018881A2 WO2000018881A2 (en) 2000-04-06
WO2000018881A3 true WO2000018881A3 (en) 2000-11-23
WO2000018881A8 WO2000018881A8 (en) 2001-04-12
WO2000018881A9 WO2000018881A9 (en) 2002-08-22

Family

ID=22289725

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/022776 WO2000018881A2 (en) 1998-09-30 1999-09-30 Intein mediated peptide ligation

Country Status (5)

Country Link
EP (1) EP1117693B1 (en)
JP (1) JP4749548B2 (en)
CA (1) CA2344764C (en)
DE (1) DE69931382T2 (en)
WO (1) WO2000018881A2 (en)

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7192596B2 (en) * 1996-08-23 2007-03-20 The Health Protection Agency Ipsen Limited Recombinant toxin fragments
DE60040524D1 (en) * 1999-02-12 2008-11-27 New England Biolabs Inc INTEIN-MEDIATED LIGATION OF EXPRESSED PROTEINS
WO2001012820A1 (en) * 1999-08-17 2001-02-22 Health Research Institute Genetic system and self-cleaving inteins derived therefrom, bioseparations and protein purification employing same, and methods for determining critical, generalizable amino acid residues for varying intein activity
DE60033551T2 (en) 2000-07-26 2007-11-08 Japan Science And Technology Agency, Kawaguchi PROBE FOR ANALYZING PROTEIN-PROTEIN INTERACTIONS AND CORRESPONDING METHOD THEREFOR
TWI353991B (en) * 2003-05-06 2011-12-11 Syntonix Pharmaceuticals Inc Immunoglobulin chimeric monomer-dimer hybrids
US7348004B2 (en) 2003-05-06 2008-03-25 Syntonix Pharmaceuticals, Inc. Immunoglobulin chimeric monomer-dimer hybrids
AU2004239244C1 (en) 2003-05-06 2015-04-23 Bioverativ Therapeutics Inc. Clotting factor-Fc chimeric proteins to treat hemophilia
CA2534520C (en) * 2003-08-05 2014-04-08 Css-Albachem Limited Oligopeptide ligation methods comprising an activated ester moiety or an aldehyde or ketone moiety
US7235589B2 (en) * 2004-09-21 2007-06-26 Bio Numerik Pharmaceuticals, Inc. Method of treating patients undergoing kidney dialysis
EP2085403A4 (en) * 2006-10-06 2012-12-19 Univ Tokai Educational System Process for production of peptide thioester
CA2885931C (en) 2012-10-03 2023-01-10 Agrivida, Inc. Intein-modified proteases, their production and industrial applications
JP2016504417A (en) 2013-01-11 2016-02-12 ザ テキサス エー アンド エム ユニヴァーシティー システムＴｈｅ Ｔｅｘａｓ Ａ＆Ｍ Ｕｎｉｖｅｒｓｉｔｙ Ｓｙｓｔｅｍ Intein-mediated protein purification
CN105263509A (en) 2013-05-31 2016-01-20 诺和诺德股份有限公司 Methods for producing peptides using engineered inteins

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834247A (en) * 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5834247A (en) * 1992-12-09 1998-11-10 New England Biolabs, Inc. Modified proteins comprising controllable intervening protein sequences or their elements methods of producing same and methods for purification of a target protein comprised by a modified protein

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHONG S. ET AL.: "Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element", GENE, vol. 192, no. 2, 19 June 1997 (1997-06-19), pages 271 - 281, XP002925637 *
EVANS T.C. ET AL.: "Semisynthesis of cytotoxic proteins using a modified protein splicing element", PROTEIN SCIENCE, vol. 7, no. 11, 5 November 1998 (1998-11-05), pages 2256 - 2264, XP002925638 *
EVANS T.C. ET AL.: "The Cyclization and Polymerization of Bacterially Expressed Proteins Using Modified Self-splicing Inteins", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 26, June 1999 (1999-06-01), pages 19359 - 18363, XP002925640 *
MUIR T.W. ET AL.: "Expressed protein ligation: A general method for protein engineering", PROC. NATL. ACAD. SCI. USA, vol. 95, June 1998 (1998-06-01), pages 6705 - 6710, XP002925639 *
See also references of EP1117693A4 *
TELENTI A. ET AL.: "The mycobacterium xenopi GyrA Protein Splicing Element: Characterization of a Minimal Intein", JOURNAL OF BACTERIOLOGY, vol. 179, no. 20, 14 October 1997 (1997-10-14), pages 6378 - 6382, XP002925641 *

Also Published As

Publication number Publication date
DE69931382D1 (en) 2006-06-22
JP4749548B2 (en) 2011-08-17
CA2344764C (en) 2011-11-29
WO2000018881A2 (en) 2000-04-06
CA2344764A1 (en) 2000-04-06
WO2000018881A8 (en) 2001-04-12
EP1117693A4 (en) 2003-06-25
JP2002526387A (en) 2002-08-20
EP1117693B1 (en) 2006-05-17
WO2000018881A9 (en) 2002-08-22
EP1117693A2 (en) 2001-07-25
DE69931382T2 (en) 2007-05-03

Similar Documents

Publication Publication Date Title
WO2000018881A3 (en) Intein mediated peptide ligation
WO2003014347A3 (en) Plant seed specific promoters
CS9100241A2 (en) Method of composite proteins' enzymic splitting
CA2061382A1 (en) Method for producing cysteine-free peptide
AU7566396A (en) Process for the preparation of peptides by way of streptavidin fusion proteins
AU5879990A (en) Stabilized fgf composition and production thereof
HK1036475A1 (en) Erythropoietin analog-human serum albumin fusion protein
EP1908773B8 (en) Process for producing dipeptides
WO2002061119A1 (en) Compositions for assaying glycoprotein
EP0330451A3 (en) A cdna coding for human normal serum albumin a, and a process for production of the albumin
GB8314362D0 (en) Polypeptide and protein products
WO2002026933A3 (en) Isoprenoid production
ZA888396B (en) Process for the production of biodegradable microcapsules of water-soluble peptides and proteins,and microcapsules obtained by this process
YU62000A (en) Process for producing the unsaturated 4,5-alleneketone, 3,5-dieneketone and corresponding saturated ketones
WO1999021830A8 (en) Process for stereoselective synthesis of prostacyclin derivatives
MX9701614A (en) Method for the production of recombinant proteins, plasmids and modified cells.
AU6474599A (en) Process for the manufacture of alpha,alpha-branched carboxylic acids
EP0867442A3 (en) A process for treating methylchlorosilanes by-products
WO2000018909A3 (en) NUCLEIC ACIDS AND PROTEINS FROM $i(CENARCHAEUM SYMBIOSUM)
WO2002043913A8 (en) Method for connecting metallic objects
AU2001241125A1 (en) Process for producing polypeptide having disulfide bond
EP1076097A4 (en) METHOD FOR CONTROLLING CLEAVAGE BY OmpT PROTEASE
EP1127958A3 (en) Process for coating a surface
ZA996751B (en) Process for the fermentative production of L-amino acids.
EP1044688A4 (en) Method for solubilizing pyridonecarboxylic acid, solubilizer therefor, aqueous solution preparations containing pyridonecarboxylic acid and process for producing the same

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C2

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: C2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: PAGES 1/11-11/11, DRAWINGS, REPLACED BY NEW PAGES 1/11-11/11; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

AK Designated states

Kind code of ref document: A3

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 572329

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref document number: 2344764

Country of ref document: CA

Ref country code: CA

Ref document number: 2344764

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1999950063

Country of ref document: EP

AK Designated states

Kind code of ref document: C1

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: C1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

CFP Corrected version of a pamphlet front page

Free format text: UNDER (57) PUBLISHED ABSTRACT REPLACED BY CORRECT ABSTRACT

WWE Wipo information: entry into national phase

Ref document number: 09786009

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1999950063

Country of ref document: EP

AK Designated states

Kind code of ref document: B9

Designated state(s): CA JP US

AL Designated countries for regional patents

Kind code of ref document: B9

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

COP Corrected version of pamphlet

Free format text: PAGES 1/11-11/11, DRAWINGS, REPLACED BY NEW PAGES 1/11-11/11; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

WWG Wipo information: grant in national office

Ref document number: 1999950063

Country of ref document: EP