Expression and purification of BC4707-RGSHis₆.

<p>Protein expression was induced for 3 hours by addition of IPTG (1 mM) to exponentially growing bacteria. Cells were harvested and mixed membrane preparations were made from each culture. The proteins in the mixed membrane fractions prepared by the water-lysis technique <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036720#pone.0036720-Ward2" target="_blank">[47]</a> were separated by SDS-PAGE and detected by: (A) Coomassie Blue stain; or (B) Western blotting with an antibody to the RGSHis₆-tag (+ and − represent mixed membrane preparations of induced and uninduced cells, respectively). Purification of BC4707-RGSHis₆ was done by immobilized metal affinity chromatography (IMAC). Staining with Coomassie Blue (C) shows the enrichment and purity of BC4707-RGSHis₆ at different stages of purification. The gel was loaded as follows: 1) molecular weight marker 2) inner membranes solubilized in dodecyl-beta-D-maltoside detergent; 3) supernatant of the solubilized inner membranes after ultracentrifugation; 4) pellet of the solubilized inner membrane fraction after ultracentrifugation; 5) flow-through after IMAC-binding; 6) elution fraction (5 µg protein). Western blot (D) of the final elution fraction (7.5 µg protein). The sizes of the bands in the molecular weight markers are indicated.