Fig. 2
Expression, optimization, and purification of CeA peptide with AT-HIS system in E. coli. Competent E. coli BL21(DE3) cells were transformed with plasmid pET21–CeA–Mxe–His, which produced the CeA–Mxe–His fusion. Cells carrying plasmid pET21–CeA–Mxe–His were cultured in LB medium to express the fusion. To induce high expression, different temperatures (16 °C, 25 °C, or 37 °C) (A) and a series of IPTG concentrations (B) were used. Different concentrations of DTT (40–80 mM) were tested to optimize the efficiency of Mxe GyrA intein cleavage (C). High-purity CeA peptide was obtained after nickel ion affinity chromatography (D). Red arrow indicates CeA–Mxe–His fusion; NO, the cells were not induced with IPTG; W, whole-cell lysate; S, supernatant fractions; and P, insoluble pellets of induced cells after sonication. “a” and “b” in C indicate the sample before and after cleavage by DTT, respectively. In D: SL, supernatant before it was loaded onto the column; ET, elution fraction; FF, fast-flow fraction; CeA, final concentrated CeA peptide. M, M1 and M2, protein markers of different molecular weights