title: V antigen-polyhistidine fusion peptide: binding to LcrH and active immunity against plague.
abstract: The structural gene for V antigen (lcrV) is known to be encoded within the lcrGVH-yopBD operon of the approximately 70-kb low-calcium-response or Lcr plasmid of Yersinia pestis. This 37-kDa monomeric peptide was reported to provide active immunity in mice, suppress inflammatory cytokines, and regulate expression of the low calcium response (Lcr+). Here we describe pVHB62, encoding a polyhistidine-V antigen fusion peptide (Vh) and linked LcrH. Vh underwent degradation from both the C terminus and N terminus during classical chromatographic fractionation but remained intact within two compartments during Ni2+ affinity chromatography. The first was homogeneous, capable of active immunization (mouse intravenous 50% lethal dose, > 10(7) bacteria), and stable at 4 degrees C. The second remained bound to the affinity column but could be eluted as a mixture of Vh, LcrH, and low-molecular-weight material by application of 6 M guanidine HCl. This mixture was dialyzed, denatured in 8 M urea, and again applied to the affinity column, which then hound Vh but not LcrH. The latter was recovered and renatured, and low-molecular-weight material was removed by biochemical fractionation. The resulting homogeneous LcrH bound protein AN antigen fusion peptide but not protein A in a sandwich enzyme-linked immunosorbent assay, and this reaction was inhibited by Vh. These observations indicate that LcrH normally binds V antigen in bacterial cytoplasm and suggest that only free LcrH down-regulates expression of the low calcium response.
authors.affiliation: Department of Microbiology, Michigan State University, East Lansing 48824-1101, USA.
authors.forename: V L
authors.initials: VL
authors.lastname: Motin
authors.forename: Y A
authors.initials: YA
authors.lastname: Nedialkov
authors.forename: R R
authors.initials: RR
authors.lastname: Brubaker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000276:immunology*; D001428:Bacterial Vaccines / Q000276:immunology*; D001483:Base Sequence; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006639:Histidine; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D010930:Plague / Q000276:immunology*; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins / Q000276:immunology* / Q000302:isolation & purification; D014614:Vaccines, Synthetic / Q000276:immunology*; D015010:Yersinia pestis / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D001428:Bacterial Vaccines; C043908:LcrV protein, Yersinia; D018832:Molecular Chaperones; D010455:Peptides; D052899:Pore Forming Cytotoxic Proteins; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; C033223:polyhistidine; D006639:Histidine