High-Level Expression and Purification of an Analgesic Peptide from Buthus martensii Karch

Abstract

BmK AngM1, a scorpion peptide isolated from Buthus martensii Karch was reported to exhibit potential analgesic effect. But the relative low content of this toxin in crude venom limits its further characterization. In this study, we constructed an expression vector and transformed into E.coli. The BmK AngM1 was expressed as a fusion protein in the soluble fraction and was purified by Nickel affinity chromatography. Subsequently, the purified fusion protein was cleaved by enterokinase and was further purified by reverse-phase HPLC. We purified 25 mg recombinant BmK AngM1 (rBmK AngM1) from 1 L bacterial culture. The molecular weight of rBmK AngM1 determined by ESI-MS was 7240.4 Da which was the expected size for correctly processed. Analgesic bioassay studies of rBmK AngM1 exhibited its potential analgesic effect comparable to that of the natural BmK AngM1 peptide.

Expression, purification and characterization of an analgesic peptide from Buthus martensii Karsch in Pichia pastoris

“…Similar results were obtained in our experiment. The production of rBmK AngM1 in P. pastoris was above 500 mg/l, which was more than three times of that in E. coli (Cao et al 2007). Bioactivity assay indicated that the analgesic effect of rBmK AngM1 expressed in P. pastoris was slightly less than that of the natural toxin but much better than that expressed in E. coli.…”

“…Therefore, it is important to develop new strategies for BmK AngM1 production. The previous attempt was made in Escherichia coli to obtain recombinant BmK AngM1 (rBmK AngM1) (Cao et al 2007). But the inconvenient purification and relatively low production of rBmK AngM1 from E. coli limited its application.…”

Strategies for Optimizing the Production of Proteins and Peptides with Multiple Disulfide Bonds

“…This strain produced high yields of heterologous secreted proteins [ 49 ]. Heterologous proteins developed as inclusion bodies in E. coli are inert, composite, and insoluble and typically exhibit non-native intra- and inter-molecular disulfide bonds and rare free cysteines [ 50 ]. Such bodies must be separated from the cell to derive the active protein along with the proteins solubilized by denaturants, and disulfide bonds must be extracted using reduction agents.…”

“…Thus, affinity tags are usually chosen depending on qualitative knowledge. To resolve this absence of a standardized analysis for affinity tags, we have previously measured the quality, yield, and expense by using two protein fusion and six short peptide affinity tags to purify two E. coli -expressed proteins [ 50 ]. We evaluated the capacity of the affinity resins to purify labeled proteins from extracts derived from yeast, Drosophila , and HeLa cells.…”