Purification and characterization of the major surface array protein from the avirulent Bacillus anthracis Delta Sterne-1.
Many prokaryotic organisms possess surface layer (S-layer) proteins that are components of the outermost cell envelope. With immunogold labeling, it was demonstrated that the protein extractable antigen 1 (EA1) was localized on the outer surface and specifically to cell wall fragments from Bacillus anthracis which retained the S layer. When grown in rich medium under aerobic conditions, the avirulent strain Delta Sterne-1 released large amounts of EA1 into the medium. This EA1 had no higher-order structure initially but formed two-dimensional crystals under defined conditions. The released EA1 was purified in aqueous buffers with a three-step procedure and found to have a mass of 95 kDa when subjected to denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). N-terminal sequence data revealed exact identity to the first eight residues of the S-layer protein from B. thuringiensis 4045. Gel permeation chromatography of the purified EA1 under nondenaturing conditions revealed a single peak corresponding to a mass of approximately 400 kDa, suggesting that a tetramer or dimer of dimers was the primary species in solution. SDS-PAGE of EA1 purified in the absence of protease inhibitors revealed specific proteolytic processing to an 80-kDa form, which immunoreacted with polyclonal anti-EA1 antibodies. This proteolytic cleavage of EA1 to 80 kDa was duplicated with purified EA1 and the protease trypsin or pronase.
Farchaus
J W
JW
Bacteriology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011, USA.
Ribot
W J
WJ
Downs
M B
MB
Ezzell
J W
JW
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001408:Bacillus anthracis / Q000737:chemistry* / Q000472:pathogenicity / Q000648:ultrastructure; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D002473:Cell Wall / Q000737:chemistry / Q000648:ultrastructure; D008562:Membrane Glycoproteins; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014774:Virulence
D000596:Amino Acids; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins