Purification of streptococcal protein g expressed byEscherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin
- 1 January 1987
- journal article
- research article
- Published by Wiley in Biomedical Chromatography
- Vol. 2 (5) , 221-225
- https://doi.org/10.1002/bmc.1130020510
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 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.Keywords
This publication has 15 references indexed in Scilit:
- Streptococcal protein G, expressed by streptococci or by Escherichia coli, has separate binding sites for human albumin and IgGMolecular Immunology, 1987
- Purification of hormone-sensitive lipase by high-performance ion exchange chromatographyAnalytical Biochemistry, 1986
- Detection and purification of rat and goat immunoglobulin G antibodies using protein G-based solid-phase radioimmunoassaysJournal of Immunological Methods, 1986
- Extraction and characterization of IgG Fc receptors from group C and group G streptococciMolecular Immunology, 1986
- Detection of receptors for the Fc region of IgG on streptococciJournal of Immunological Methods, 1983
- Immobilization of enzymes and affinity ligands to various hydroxyl group carrying supports using highly reactive sulfonyl chloridesBiochemical and Biophysical Research Communications, 1981
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Analysis of cysteinyldopas, dopa, dopamine, noradrenaline and adrenaline in serum and urine using high-performance liquid chromatography and electrochemical detectionJournal of Chromatography B: Biomedical Sciences and Applications, 1979
- High performance liquid affinity chromatography (HPLAC) and its application to the separation of enzymes and antigensFEBS Letters, 1978
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970