FIG. 6.

VapC-1 was successfully purified via tandem cloning into pET24b and expression in BL21(DE3). (A) The vapBC-1 operon was fused to pET24b such that vapB-1 was in frame with the vector's ATG start codon at the N terminus, and vapC-1 was in frame with the C-terminal polyhistidine tag, creating pDD686. Induction of the construct with IPTG resulted in no significant growth inhibition of the expression strain. (B) Coomassie-stained 12% SDS-PAGE separation of a typical purified pET::VapC-1 construct (3.5 μg). Two bands are visible: one at the calculated molecular mass of pET::VapC-1 (16.6 kDa) and one at the size of pET::VapB-1 (10.5 kDa). (C) Identical immunoblot probed with anti-His C-terminal HRP-linked monoclonal antibody. Note that only a single band is apparent.