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Figure 3 | BMC Biotechnology

Figure 3

From: Production and purification of polymerization-competent HIV-1 capsid protein p24 (CA) in NiCo21(DE3) Escherichia coli

Figure 3

Optimization of IPTG concentration and induction temperature for the expression of HIV-1 CA. Determination of optimal inducer concentration: The pSA-Hp24-6His-transformed NiCo21 (DE3) E. coli cultures (0.6OD600) were added with varying concentrations (0–0.4 mM) of IPTG and incubated at 30°C for 6 hours. Eight micro liter of cultures were mixed with 4X loading dye, boiled, and electrophoresed on 12% gels. Proteins were transferred to NC membranes and immunoblot analysis was carried out as described in Methods. (A). Determination of optimal induction temperature: The pSA-Hp24-6His-transformed NiCo21(DE3) E.coli cultures (0.6OD600) were induced with 0.05 mM IPTG and incubated at 30, 22, and 18°C for 6, 12, and 18 hours respectively. Cultures were then processed to obtain insoluble (IS) and soluble (S) fractions and electrophoresed as described in Methods. Gels were stained with Coomassie Blue G250 and photographed (B). Lane M1, BenchMark Pre-stained protein ladder; Lane 1: Un-induced culture at 30°C (soluble); Lane 2, IPTG-induced culture at 30°C (insoluble); Lane 3, IPTG-induced culture at 30°C (soluble); Lane 4: Un-induced culture at 22°C (soluble); Lane 5, IPTG-induced culture at 22°C (insoluble); Lane 6, IPTG-induced culture at 22°C (soluble); Lane 7: Un-induced culture at 18°C (soluble); Lane 8, IPTG-induced culture at 18°C (insoluble); Lane 9, IPTG-induced culture at 18°C (soluble).

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