V antigen-polyhistidine fusion peptide: binding to LcrH and active immunity against plague

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.