Soluble prokaryotic expression and purification of crotamine using an N-terminal maltose-binding protein tag

In our current study, we show for the first time that N-terminal fusion with three protein tags-N-utilization substance protein A (NusA), protein disulfide isomerase b′a′ domain (PDIb′a′), and maltose-binding protein (MBP)-enables the soluble overexpression of crotamine in the cytoplasm of Escherichia coli. MBP-tagged crotamine was purified using Ni affinity, anion exchange, and MBP chromatography. The tag was cleaved using TEV protease, and the final product was pure on a silver-stained gels. In total, 0.9 mg pure crotamine was obtained from each liter of bacterial culture with endotoxin level approximately 0.15 EU/μg, which is low enough to use in biomedical applications. The identity and intramolecular disulfide bonds were confirmed using MALDI-TOF MS analysis. Purified crotamine inhibited the hKv1.3 channel (but not hKv1.5) in a dose-dependent manner with IC50 value of 67.2 ± 44.7 nM (n = 10), indicating the correct protein folding. The crotamine product fused with MBP at its N-terminus also inhibited the hKv1.3 channel, suggesting that the N-terminus is not involved in the channel binding of the toxin.