Optimization of IPTG concentration and induction temperature.
(A) For optimization of IPTG concentration, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with varying concentrations (0, 0.05, 0.1, 0.2, 0.3, and 0.4 mM) of IPTG and grown for another 6 h at 30°C. Nine microliters of samples were mixed with 4x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by immuno-blotting. A concentration of 0.05 mM IPTG (red arrow) was sufficient to induce high level Nef expression. (B) For optimal induction temperature, the overnight cultures of pSA-HNef-6His-transformed NiCo21(DE3) were diluted 1:100 in LB+Amp (100 μg/ml) and grown to mid-log phase (OD600 ~0.5–0.6). The cultures were then induced with 0.05 mM of IPTG and grown at 30, 22, and 18°C for 6, 12, and 16 h respectively. Nine microliters of samples were mixed with 4 x loading dye, electrophoretically resolved on a 12% SDS-PAGE gel and analyzed by Coomassie staining. Nef expression was similar at 30 and 22°C but lower at 18°C. Lanes: M, BenchMark Pre-stained protein ladder; M1, PageRuler Prestained Protein Ladder Plus; 1, un-induced NiCo21 at 30°C (soluble); 2, IPTG-induced NiCo21 at 30°C (insoluble); 3, IPTG-induced NICo21 at 30°C (soluble); 4, un-induced NiCo21 at 22°C (soluble); 5, IPTG-induced NiCo21 at 22°C (insoluble); 6, IPTG-induced NiCo21 at 22°C (soluble); 7, un-induced NiCo21 at 18°C (soluble); 8, IPTG-induced NiCo21 at 18°C (insoluble); 9, IPTG-induced NiCo21 at 18°C (soluble).
