Preparation of bacterial recombinant proteins. (A) Expression. Arrowheads indicate 5 recombinant proteins expressed in E coli after IPTG induction: GST-D1459R1668-H (I), GST-E1554R1668-H (II), GST-D1587R1668-H (III), GST-D1596R1668-H (IV), and GST-D1596R1659-H (V). Gels after SDS-PAGE were stained with GelCode Blue (Pierce, Rockford, IL). The sizes of the protein standards are indicated at the left. (B) Fractionation. Recombinant proteins I and II were collected in pellet fractions (P) after centrifugation, whereas III, IV, and V were in soluble fractions (S). (C) Purification. All the recombinant proteins were purified by 2 sequential column-chromatography procedures, nickel-ion chelating chromatography and glutathione-affinity chromatography. The representative pattern of GST-D1596R1668-H is shown. S indicates soluble fraction of bacterial lysate; F1, flow-through of nickel-ion column; W1, wash; E1, eluate; F2, flow-through of glutathione column; W2, wash; and E2, eluate.