Purification of streptococcal protein G expressed by Escherichia coli by high performance liquid affinity chromatography using immobilized immunoglobulin G and albumin.
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.
Falkenberg
C
C
Department of Physiological Chemistry 3, University of Lund, Sweden.
Björck
L
L
Akerström
B
B
Nilsson
S
S
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D006801:Humans; D007074:Immunoglobulin G; D011863:Radioimmunoassay; D011994:Recombinant Proteins; D012709:Serum Albumin
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D011994:Recombinant Proteins; D012709:Serum Albumin