Figure 1

Construction of plasmid and Optimization of expression condition. (A) pGEX-6P-1 vector map. (B-C) Effect of temperature on GST-DUSP5 protein expression. (B) Lane 1: MW marker, lanes 2, 4, 6 and 8: cell pellet before induction, lanes 3, 5, 7 and 9: cell pellet after induction. Lanes 2–3, 4–5 are induced at 25°C for 20 h, lanes 6–7, 8–9 are induced at 16°C for 20 h. Lanes 4–5 and 8–9 are duplicate samples for lanes 2–3 and 4–5 respectively. (C) Effect of temperature on GST-DUSP5 solubility, Lane 1: MW marker, lanes 2 & 4 and 3 & 5 are cell pellet, and cell lysate post sonication, and samples induced at 25°C overnight for 20 h, lanes 6 & 8 and 7 & 9 are cell pellet and cell lysate post sonication, and samples induced at 16°C overnight for 20 h. (D-E) Effect of IPTG concentration on GST-DUSP5 protein expression. (D) Lane 1: MW marker, lanes 2, 4, 6 & 8: cell pellet before induction, and lanes 3, 5, 7 & 9: cell pellet after induction either with 0.01 mM or 0.05 mM IPTG at 25°C or 16°C for 20 h. (E) Effect of IPTG concentration on GST-DUSP5 solubility. Lane 1: MW marker, lanes 2, 4, 6 & 8 cell pellet post sonication, and lanes 3, 5, 7 & 9 are cell lysate post sonication for expression induced with either 0.01 mM or 0.05 mM IPTG, at 25°C or 16°C. (F) GST-DUSP5 protein expression using terrific broth and 0.05 mM and 0.1 mM IPTG and 16°C overnight induction. Lane 1: MW marker, lanes 2 & 4 and 3 & 5 are cell pellet before and after induction. Lanes 2–3: 0.05 mM IPTG, lanes 4–5: 0.1 mM IPTG. (G) GST-DUSP5 protein solubility and GST pull down. Lane 1: MW marker, lanes 2 & 5 are cell pellet post sonication, lanes 3 & 6 are cell lysate post sonication, lanes 4 & 7 are protein adsorbed on GST beads.