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Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin

Falkenberg, C LU ; Björck, L LU ; Åkerström, B LU and Nilsson, S LU (1987) In Biomedical Chromatography 2(5). p.5-221
Abstract

A one-step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G- and albumin-binding properties. Lysed Escherichia coli bacteria infected with lambda-phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750-fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45... (More)

A one-step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G- and albumin-binding properties. Lysed Escherichia coli bacteria infected with lambda-phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750-fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45 and 47 kilodaltons, respectively. A specific method for quantitation of small amounts of protein G was developed and used for specific tracing of the protein after the affinity chromatography. Goat polyclonal antibodies were bound to an antigen coated to the plastic walls of microtiter plates, causing the Fc-region of the immunoglobulins to be directed outwards. Unknown samples of protein G were then allowed to compete with radio-iodinated protein G (solid phase radioassay) or protein G coupled to alkaline phosphatase (enzyme linked sorbent assay) for the Fc-regions.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Bacterial Proteins/isolation & purification, Chromatography, Affinity, Chromatography, High Pressure Liquid, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Humans, Immunoglobulin G, Radioimmunoassay, Recombinant Proteins, Serum Albumin
in
Biomedical Chromatography
volume
2
issue
5
pages
5 - 221
publisher
John Wiley & Sons
external identifiers
  • scopus:0023625021
  • pmid:3333727
ISSN
0269-3879
DOI
10.1002/bmc.1130020510
language
English
LU publication?
yes
id
95056a70-435a-4fa1-9985-907d0a2ce4d3
date added to LUP
2019-05-22 10:32:33
date last changed
2020-01-13 01:51:57
@article{95056a70-435a-4fa1-9985-907d0a2ce4d3,
  abstract     = {<p>A one-step HPLC method was developed for the purification of protein G, a cell wall molecule from group C and G streptococci with immunoglobulin G- and albumin-binding properties. Lysed Escherichia coli bacteria infected with lambda-phages containing the protein G gene from group G streptococci were used as a starting material for the preparations. The lysate was applied to a column with immobilized human immunoglobulin G or human serum albumin. Protein G was selectively bound and eluted at pH 2.0. A 750-fold purification was achieved. Sodium dodecylsulfate + polyacrylamide gel electrophoresis showed that the highly purified protein G consisted of three sets of doublets with the apparent molecular weight of 64 and 67, 56 and 58, and 45 and 47 kilodaltons, respectively. A specific method for quantitation of small amounts of protein G was developed and used for specific tracing of the protein after the affinity chromatography. Goat polyclonal antibodies were bound to an antigen coated to the plastic walls of microtiter plates, causing the Fc-region of the immunoglobulins to be directed outwards. Unknown samples of protein G were then allowed to compete with radio-iodinated protein G (solid phase radioassay) or protein G coupled to alkaline phosphatase (enzyme linked sorbent assay) for the Fc-regions.</p>},
  author       = {Falkenberg, C and Björck, L and Åkerström, B and Nilsson, S},
  issn         = {0269-3879},
  language     = {eng},
  number       = {5},
  pages        = {5--221},
  publisher    = {John Wiley & Sons},
  series       = {Biomedical Chromatography},
  title        = {Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin},
  url          = {http://dx.doi.org/10.1002/bmc.1130020510},
  doi          = {10.1002/bmc.1130020510},
  volume       = {2},
  year         = {1987},
}