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WO1998001560A1 - Proteine g modifiee et fragments de ladite proteine - Google Patents

Proteine g modifiee et fragments de ladite proteine Download PDF

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Publication number
WO1998001560A1
WO1998001560A1 PCT/GB1997/001818 GB9701818W WO9801560A1 WO 1998001560 A1 WO1998001560 A1 WO 1998001560A1 GB 9701818 W GB9701818 W GB 9701818W WO 9801560 A1 WO9801560 A1 WO 9801560A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide sequence
mutation
protein
fab
fragments
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/GB1997/001818
Other languages
English (en)
Inventor
Jeremy Derrick
Gordon Roberts
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MANCHESTER INSTITUTE OF SCIENCE, University of
MANCHESTER INSTITUTE OF SCIENCE and TECHNOLOGY, University of
University of Leicester
Original Assignee
MANCHESTER INSTITUTE OF SCIENCE, University of
MANCHESTER INSTITUTE OF SCIENCE and TECHNOLOGY, University of
University of Leicester
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 MANCHESTER INSTITUTE OF SCIENCE, University of, MANCHESTER INSTITUTE OF SCIENCE and TECHNOLOGY, University of, University of Leicester filed Critical MANCHESTER INSTITUTE OF SCIENCE, University of
Priority to AU34513/97A priority Critical patent/AU3451397A/en
Priority to GB9828306A priority patent/GB2333295A/en
Publication of WO1998001560A1 publication Critical patent/WO1998001560A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C07K16/065—Purification, fragmentation

Definitions

  • a modified protein G, or fragment thereof in accordance with the first aspect of the invention, is useful for effecting separation of the Fab or Fc fragment of an antibody from a mixture thereof.
  • the selectivity of binding displayed by the polypeptide sequences of the invention (as compared to unmodified protein G) may be expressed m terms o :
  • the preferred mutation is at position 40 (according to the nomenclature of Fig. 1 ) of domain I, II or III, the most preferred mutation being the N40A mutation (i.e. replacement of asparagine at position 40 by alanine).
  • 2YT medium was prepared as described by Sambrook et al. ( 1 89). 1 .8 Buffers
  • the PCR incubation was prepared as follows
  • the TrisHCl/MgCl2 solution was steiilised by autoclaving and DTT added subsequently.
  • the ATP and dNTP solutions were steiilised by filtration.
  • 10 ⁇ l of the polymerase/ligase solution was added to the annealed template and primer (to give 20 ⁇ l total volume) and incubated at 1 5 °C toi 15 houis 80 ⁇ l ol sienle water was added to the incubation and 5 ⁇ l was used to tianstorm E coli .IM1 1 b ⁇ the CaCb method (Maniatis et al 1982) l iansfoimants containing mpl 8 JP l with the appropriate N40A mutation were identified by dideoxy DNA sequencing of the sequence between the BamHI and Hind III sites of mpl 8 JPD l .
  • a microtitre plate-based assay was used to measuie the lelative binding affinities of the wild type and mutant piotein G domains toi Fab and fc fragments.
  • a 96 well plastic microtitre plate was coated overnight with Fab or Fc fragment in 15mM Na 2 C0 3 /35mM NaHCO , at loom temperatuie
  • piotein concentiations ⁇ j 10 ⁇ g/ml lor Fab and 35 ⁇ g'ml foi l c fragments were used.
  • PBS phosphate buffered saline solution
  • Tween-20 0.05% Tween-20.
  • PBS contains 0.01 M NaH 2 P0 4 NaOH (pH 7.4), 0.138M NaCI and 2.7mM KG.
  • the relative binding affinity of each protein G domain was assessed by competition for adhesion to the plate with an alkaline phosphatase-protein G conjugate, prepared as described below.
  • the alkaline phosphatase-protein G conjugate was diluted 1000-fold into PBS/0.05% Tween-20 to give a final protein concentration of approximately lO ⁇ g/ml, and a variable amount of competing protein G domain was added (generally to a final concentration between 5mg/ml and 5ug/ml).
  • the protein G domain wild type or mutant was purified as previously described.
  • the alkaline phosphatase-protein G conjugate was then incubated within the microtitre plate well for 90 minutes, during which time it bound to Fab or Fc components that were immobilised on the plate.
  • the slitwidth of excitation used was 15 nm. the slitwidth of emission 10 nm. the excitation wavelength 337 nm. the emission wavelength 480 nm and the temperature 298 K.
  • the concentration of human IgG was 62.9 nM and T23C- AEDANS was 131.5 nM.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Une séquence polypeptidique se présentant sous la forme d'une protéine G modifiée ou d'un fragment de ladite protéine se fixe sélectivement à l'un ou l'autre des fragments Fab ou Fc d'un anticorps, comparé à la sélectivité de liaison correspondante d'une protéine G non modifiée.
PCT/GB1997/001818 1996-07-04 1997-07-04 Proteine g modifiee et fragments de ladite proteine Ceased WO1998001560A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU34513/97A AU3451397A (en) 1996-07-04 1997-07-04 Modified protein g and fragments thereof
GB9828306A GB2333295A (en) 1996-07-04 1997-07-04 Modified protein G and fragments thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB9614033.0A GB9614033D0 (en) 1996-07-04 1996-07-04 Modified protein G and fragments thereof
GB9614033.0 1996-07-04

Publications (1)

Publication Number Publication Date
WO1998001560A1 true WO1998001560A1 (fr) 1998-01-15

Family

ID=10796342

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1997/001818 Ceased WO1998001560A1 (fr) 1996-07-04 1997-07-04 Proteine g modifiee et fragments de ladite proteine

Country Status (3)

Country Link
AU (1) AU3451397A (fr)
GB (1) GB9614033D0 (fr)
WO (1) WO1998001560A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003088381A (ja) * 2001-09-18 2003-03-25 Kanegafuchi Chem Ind Co Ltd 新規ペプチド、生産方法、新規吸着体、吸着器および吸着方法
WO2008052933A3 (fr) * 2006-10-30 2008-11-13 Domantis Ltd Nouveaux polypeptides et leurs utilisations
JP2011050272A (ja) * 2009-08-31 2011-03-17 Univ Of Ryukyus 分子量マーカー及び分子量マーカーの作製方法
EP2740792A4 (fr) * 2011-08-04 2015-02-18 Nat Inst Of Advanced Ind Scien Nouvelle protéine modifiée comportant un multimère de type tandem d'un domaine extracellulaire mutant de protéine g
WO2015030094A1 (fr) * 2013-08-30 2015-03-05 株式会社カネカ PEPTIDE SE LIANT À LA RÉGION Fab
WO2015103545A1 (fr) * 2014-01-03 2015-07-09 Bio-Rad Laboratories, Inc. Élimination des impuretés des éluats de la protéine a
WO2016031909A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ Peptide se liant à l'immunoglobuline g
WO2016031926A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ Peptide se liant à la région fab
WO2016031902A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ MATRICE DE SÉPARATION PAR AFFINITÉ POUR UN PEPTIDE CONTENANT UNE RÉGION Fab
WO2016061427A1 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Procédés et compositions impliquant des variants de la protéine g
US10428120B2 (en) 2011-09-23 2019-10-01 Universitat Stuttgart Serum half-life extension using IgBD

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DERRICK J.P. AND WIGLEY D.B.: "Analysis of bacterial immunoglobulin-binding proteins by X-ray crystallography", IMMUNOMETHODS, vol. 2, no. 1, 1 February 1993 (1993-02-01), pages 9 - 15, XP002045028 *
DERRICK J.P. AND WIGLEY D.B.: "The third IgG-binding domain from streptococcal protein G", JOURNAL OF MOLECULAR BIOLOGY, vol. 243, 1994, pages 906 - 918, XP002045033 *
ERNTELL M. ET AL.: "Streptococcal protein G has affinity for both Fab- and Fc-fragments of human IgG", MOLECULAR IMMUNOLOGY, vol. 25, no. 2, 1988, pages 121 - 126, XP002045031 *
GOWARD C.R. ET AL.: "Expression and purification of a truncated recombinant streptococcal protein G", THE BIOCHEMICAL JOURNAL, vol. 267, no. 1, 1 April 1990 (1990-04-01), pages 171 - 177, XP002045030 *
LIAN L.-Y. ET AL.: "Determination of the solution structures of domains II and III of protein G from Streptococcus by 'H nuclear magnetic resonance", JOURNAL OF MOLECULAR BIOLOGY, vol. 228, no. 4, 1992, pages 1219 - 1234, XP002045032 *
SAUER-ERIKSSON A.E. ET AL.: "Crystal structure of the C2 fragment of streptococcal protein G in complex with the Fc domain of human IgG", STRUCTURE, vol. 3, no. 3, 15 March 1995 (1995-03-15), pages 265 - 278, XP002045029 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003088381A (ja) * 2001-09-18 2003-03-25 Kanegafuchi Chem Ind Co Ltd 新規ペプチド、生産方法、新規吸着体、吸着器および吸着方法
WO2003025011A1 (fr) * 2001-09-18 2003-03-27 Kaneka Corporation Peptide, adsorbant, unite et methode d'adsorption
WO2008052933A3 (fr) * 2006-10-30 2008-11-13 Domantis Ltd Nouveaux polypeptides et leurs utilisations
US8236931B2 (en) 2006-10-30 2012-08-07 Glaxo Group Limited Prevention of aggregation of immunoglobulin light or heavy chains
JP2011050272A (ja) * 2009-08-31 2011-03-17 Univ Of Ryukyus 分子量マーカー及び分子量マーカーの作製方法
EP2740792A4 (fr) * 2011-08-04 2015-02-18 Nat Inst Of Advanced Ind Scien Nouvelle protéine modifiée comportant un multimère de type tandem d'un domaine extracellulaire mutant de protéine g
US10428120B2 (en) 2011-09-23 2019-10-01 Universitat Stuttgart Serum half-life extension using IgBD
WO2015030094A1 (fr) * 2013-08-30 2015-03-05 株式会社カネカ PEPTIDE SE LIANT À LA RÉGION Fab
US10556944B2 (en) 2013-08-30 2020-02-11 Kaneka Corporation Fab region-binding peptide
CN105518023A (zh) * 2013-08-30 2016-04-20 株式会社钟化 Fab区域结合性肽
JPWO2015030094A1 (ja) * 2013-08-30 2017-03-02 株式会社カネカ Ｆａｂ領域結合性ペプチド
US10584150B2 (en) 2014-01-03 2020-03-10 Bio-Rad Laboratories, Inc. Removal of impurities from protein A eluates
WO2015103545A1 (fr) * 2014-01-03 2015-07-09 Bio-Rad Laboratories, Inc. Élimination des impuretés des éluats de la protéine a
US9546208B2 (en) 2014-01-03 2017-01-17 Bio-Rad Laboratories, Inc. Removal of impurities from protein A eluates
WO2016031902A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ MATRICE DE SÉPARATION PAR AFFINITÉ POUR UN PEPTIDE CONTENANT UNE RÉGION Fab
JPWO2016031909A1 (ja) * 2014-08-28 2017-06-15 株式会社カネカ 免疫グロブリンｇ結合性ペプチド
JPWO2016031926A1 (ja) * 2014-08-28 2017-06-22 株式会社カネカ Ｆａｂ領域結合性ペプチド
US10494414B2 (en) 2014-08-28 2019-12-03 Kaneka Corporation Immunoglobulin G-binding peptide
WO2016031926A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ Peptide se liant à la région fab
WO2016031909A1 (fr) * 2014-08-28 2016-03-03 株式会社カネカ Peptide se liant à l'immunoglobuline g
WO2016061427A1 (fr) * 2014-10-17 2016-04-21 The University Of Chicago Procédés et compositions impliquant des variants de la protéine g
US10759834B2 (en) 2014-10-17 2020-09-01 The University Of Chicago Methods and compositions involving protein G variants

Also Published As

Publication number Publication date
GB9614033D0 (en) 1996-09-04
AU3451397A (en) 1998-02-02

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