Figure 2.
Expression and solubility of PfBCCP. (A) Coomassie-stained SDS-PAGE gel showing soluble and insoluble fractions from cells expressing full length or truncated PfBCCP. The full-length 214-residue biotin carboxy carrier protein (BCCP) domain of the P. falciparum ACC gene (PfBCCP) and a 79-residue fragment containing the biotinylation site (PfBCCP-79) were expressed as GST fusion proteins in BL21-Star(DE3) cells. Whole cell lysate was separated into insoluble (pellet) and soluble (sol) fractions by centrifugation. The truncated fragment was predominantly soluble. (B) SDS-PAGE gel showing GST-tagged PfBCCP-79 (GST-BCCP) purified by affinity chromatography on a GSTrap™ Fast-Flow column (lane 1) and cleaved in vitro by tobacco etch virus (TEV) protease (lane 2). The sample was reapplied to the column to remove free GST and the purified protein (lane 3) was used for production of antisera in rabbits. PfBCCP-79 was also purified with a six-histidine tag (His-BCCP) on a metal chelating column charged with NiCl2 (lane 4).