Proteolysis of V antigen from Yersinia pestis.
Lcr-plasmids of yersiniae are known to mediate a unique low calcium response characterised by restriction of growth in vitro with induction of putative virulence factors including yersiniae outer membrane-peptides (YOPs) and V antigen (Lcr+). A medium was developed that permitted expression of high yields of V by Yersinia pestis KIM in large fermenter vessels. Immunoblots of specific precipitates prepared by prior molecular sieving showed that native unaggregated V exists as a monomeric 37,000 dalton peptide. Fractionation by precipitation with (NH4)2SO4 and chromatography on phenyl-Sepharose, DEAE cellulose, Sephacryl S200, calcium hydroxyapatite, and Sephadex G200 yielded highly purified antigen as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of parallel preparations from Lcr+ and Lcr- yersiniae. However, yields of V obtained by this process were unexpectedly low. As determined from immunoblots with monospecific polyclonal and monoclonal anti-V, this loss of activity occurred as a function of evident degradation at every step of purification yielding antigenic fragments of about 36,000, 34,000, 31,000, 30,000, and 28,000 daltons. Neutral or acidic pH favored hydrolysis; insignificant cleavage occurred in viable Lcr+ cells of Y. pestis or in culture supernatant fluids. V in neutral cytoplasm from Yersinia pseudotuberculosis or Yersinia enterocolitica did not undergo comparable degradation.
Brubaker
R R
RR
Department of Microbiology and Public Health, Michigan State University, East Lansing 48824-1101.
Sample
A K
AK
Yu
D Z
DZ
Zahorchak
R J
RJ
Hu
P C
PC
Fowler
J M
JM
D000942:Antigens, Bacterial / Q000302:isolation & purification / Q000378:metabolism*; D002845:Chromatography / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D017886:Durapatite; D006882:Hydroxyapatites; D015151:Immunoblotting; D008970:Molecular Weight; D052899:Pore Forming Cytotoxic Proteins; D015010:Yersinia pestis / Q000254:growth & development*
D000942:Antigens, Bacterial; D006882:Hydroxyapatites; C043908:LcrV protein, Yersinia; D052899:Pore Forming Cytotoxic Proteins; D017886:Durapatite