PURIFICATION OF MYCOPLASMA-GALLISEPTICUM MEMBRANE-PROTEINS P52, P67 (PMGA), AND P77 BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

The plasma membrane of the avian pathogen Mycoplasma gallisepticum con tains about 200 polypeptides including the major lipoprotein pMGA. We have developed a simple and efficient procedure for the purification o f three membrane proteins of this wall-less bacterium. Proteins were s electively extracted from isolated plasma membranes with the mild zwit terionic detergent (N-dodecyl-N,N-dimethylammonio) undecanoate (DDMAU) and subjected to size-exclusion chromatography (FPLC) in the presence of the same detergent. Two of the thus separated protein fractions we re subjected to a third step involving an anion-exchange chromatograph y (HPLC), also in the presence of DDMAU, which led to the purification to homogeneity of p67, the major acyl protein of M. gallisepticum pla sma membrane (yield, 40%; purification factor, II), p52 (yield, 38%; p urification factor, 20), and p77 (yield, approximate to 45%; purificat ion factor, 500). Analyses performed by Western blotting and crossed i mmunoelectrophoresis showed that the three purified proteins are disti nct antigens. Furthermore, N-terminal sequencing confirmed that p67 is pMGA. The method described in this paper is simple, efficient, and no ndenaturing; it provides pure proteins, at the milligram level for p52 and p67, and should prove easy to being scaled-up if necessary. (C) 1 996 Academic Press, Inc.