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 contains about 200 polypeptides including the major lipoprotein pMGA. We have developed a simple and efficient procedure for the purification of three membrane proteins of this wall-less bacterium. Proteins were selectively extracted from isolated plasma membranes with the mild zwitterionic 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 were subjected to a third step involving an anion-exchange chromatography (HPLC), also in the presence of DDMAU, which led to the purification to homogeneity of p67, the major acyl protein of M. gallisepticum plasma membrane (yield, 40%; purification factor, 11), p52 (yield, 38%; purification factor, 20), and p77 (yield, approximately 45%; purification factor, 500). Analyses performed by Western blotting and crossed immunoelectrophoresis showed that the three purified proteins are distinct antigens. Furthermore, N-terminal sequencing confirmed that p67 is pMGA. The method described in this paper is simple, efficient, and nondenaturing; it provides pure proteins, at the milligram level for p52 and p67, and should prove easy to being scaled-up if necessary.
Jan
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Centre National de la Recherche Scientifique, URA 256, Université de Rennes, France.
Brenner
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Wróblewski
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D000954:Antigens, Surface / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000302:isolation & purification; D015153:Blotting, Western; D002458:Cell Fractionation; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000494:pharmacology; D007122:Immunoelectrophoresis; D008565:Membrane Proteins / Q000302:isolation & purification*; D009174:Mycoplasma / Q000737:chemistry*; D010455:Peptides / Q000302:isolation & purification; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D017421:Sequence Analysis
D000954:Antigens, Surface; D001426:Bacterial Proteins; D003902:Detergents; D005227:Fatty Acids; D008565:Membrane Proteins; D010455:Peptides; D000644:Quaternary Ammonium Compounds; C093274:(N-dodecyl-N,N-dimethylammonio)undecanoic acid