A physicochemical study of protein G, a molecule with unique immunoglobulin G-binding properties.
Protein G, an IgG-binding molecule, was prepared from the cell walls of a group G streptococcal strain, G-148. The protein could be extracted from the cells by papain digestion and purified by the sequential use of ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Sepharose-coupled human IgG, and gel chromatography on Sephadex G-200. Two protein bands with similar molecular weight, 34,000 and 36,000, were obtained when analyzing the pure protein G on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The yield using this purification scheme was 27% of the protein G solubilized from the cells or 70 micrograms/ml packed bacteria. The Stokes radius and frictional ratio of protein G were determined to 3.53 nm and 1.64, respectively, suggesting an elongated fibrous molecule. The protein did not contain any intrachain disulfide bonds. The amino acid composition of protein G was determined and was found to be different from that of protein A, the well known staphylococcal IgG-binding protein. The equilibrium constants of the reactions between protein G and human, rabbit, mouse, and goat polyclonal IgG, determined by Scatchard plots, ranged between 1 X 10(10) and 7 X 10(10), for rat polyclonal IgG 1.4 X 10(9), and human monoclonal IgG1, IgG2, IgG3, and IgG4 between 2 X 10(9) and 6 X 10(9). These affinity constants were always greater than the corresponding values for protein A. The binding between protein G and various polyclonal and monoclonal IgG was pH dependent between 2.8 and 10, strongest at pH 4 and 5, and weakest at pH 10.
D000818:Animals; D001426:Bacterial Proteins / Q000276:immunology* / Q000302:isolation & purification; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002845:Chromatography; D006041:Goats; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D011817:Rabbits; D051381:Rats; D013291:Streptococcus / Q000032:analysis*; D013696:Temperature
D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G