Simplified method for the purification of group A streptococcal M-proteins: solution of the multiple banding problem.
A simple and rapid procedure for the isolation in high yield (about a 30% recovery based on the total 30 to 60% ammonium sulfate recovery) of homogeneous purified group A streptococcal M-protein is described. M-proteins extracted from whole cells of group A streptococci by treatment with hot HCl were neutralized, fractionated with ammonium sulfate, dialyzed, lyophilized, and then subjected to treatment with hot 60% trichloroacetic acid. This was shown to produce an M-protein preparation, free of group A carbohydrate activity and extraneous antigens, in yields up to 10-fold higher than previous methods in about one-fifth the time. These M-protein preparations were shown to: (i) have similar amino acid compositions to their respective type-specific proteins purified by diethylaminoethyl and O-(carboxymethyl) cellulose chromatography, (ii) react with their respective type-specific antisera in Ouchterlony diffusion, (iii) produce antisera in rabbits capable of promoting streptococcal long-chain formation in vitro, and (iv) give only one major band on polyacrylamide gel disk electrophoresis. The data allow for an explanation of the hitherto described multiple banding M-proteins seen on acrylamide electrophoresis.
D000596:Amino Acids / Q000032:analysis; D000645:Ammonium Sulfate; D000942:Antigens, Bacterial; D001322:Autoanalysis; D001426:Bacterial Proteins / Q000032:analysis / Q000302:isolation & purification*; D011232:Chemical Precipitation; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D005612:Freeze Drying; D006851:Hydrochloric Acid; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008722:Methods; D012703:Serotyping; D013056:Spectrophotometry, Ultraviolet; D013291:Streptococcus / Q000032:analysis*; D014238:Trichloroacetic Acid
D000596:Amino Acids; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D014238:Trichloroacetic Acid; D006851:Hydrochloric Acid; D000645:Ammonium Sulfate