Purification and partial characterization of the OmpA family of proteins of Pasteurella haemolytica.
This study was conducted to partially characterize and identify the purity of two major outer membrane proteins (OMPs) (with molecular weights of 32,000 and 35,000 [32K and 35K, respectively]) of Pasteurella haemolytica. The 35K and 32K major OMPs, designated Pasteurella outer membrane proteins A and B (PomA and PomB, respectively), were extracted from P. haemolytica by solubilization in N-octyl polyoxyl ethylene. The P. haemolytica strain used was a mutant serotype A1 from which the genes expressing the 30-kDa lipoproteins had been deleted. PomA and PomB were separated and partially purified by anion-exchange chromatography. PomA but not PomB was heat modifiable. The N-terminal amino acid sequences of the two proteins were determined and compared with reported sequences of other known proteins. PomA had significant N-terminal sequence homology with the OmpA protein of Escherichia coli and related proteins from other gram-negative bacteria. Moreover, polyclonal antiserum raised against the E. coli OmpA protein reacted with this protein. PomA was surface exposed, was conserved among P. haemolytica biotype A serotypes, and had porin activity in planar bilayers. No homology between the N-terminal amino acid sequence of PomB and those of other known bacterial proteins was found. Cattle vaccinated with live P. haemolytica developed a significant increase in serum antibodies to partially purified PomA, as shown by enzyme-linked immunosorbent assays, and to purified PomA and PomB, as detected on Western blots and by densitometry.
Mahasreshti
P J
PJ
Department of Anatomy, Pathology, and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.
Murphy
G L
GL
Wyckoff
J H
JH
Farmer
S
S
Hancock
R E
RE
Confer
A W
AW
D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D002417:Cattle; D003902:Detergents; D008051:Lipid Bilayers; D008074:Lipoproteins / Q000737:chemistry* / Q000276:immunology; D008168:Lung / Q000473:pathology; D016978:Mannheimia haemolytica / Q000737:chemistry* / Q000145:classification / Q000276:immunology; D008969:Molecular Sequence Data; D010326:Pasteurella Infections / Q000276:immunology / Q000473:pathology; D011092:Polyethylene Glycols; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012703:Serotyping; D012995:Solubility
D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D003902:Detergents; D008051:Lipid Bilayers; D008074:Lipoproteins; C103352:PomA protein, bacteria; C103353:PomB protein, bacteria; C072375:n-octyl-polyoxyethylene; D011092:Polyethylene Glycols