In vitro refolding and functional analysis of polyhistidine-tagged Buthus martensii Karsch antitumor-analgesic peptide produced in Escherichia coli.

OBJECTIVES

To identify an efficient in vitro refolding method to generate highly active His6-tagged scorpion toxin antitumor-analgesic peptide (AGAP) isolated from Escherichia coli inclusion bodies.

RESULTS

N- and C-Terminal His6-tagged recombinant (r) AGAP (NHis6-rAGAP and CHis6-rAGAP, respectively) were expressed in E. coli; the purification and refolding conditions were optimized. CHis6-rAGAP, but not NHis6-rAGAP, exhibited significant in vitro antihepatoma activity that was much greater than that of rAGAP produced using SUMO fusion technology (IC50, 0.4 ± 0.08 vs. 1.8 ± 0.3 μM). CHis6-rAGAP also showed significant inhibition of tumor growth in a mouse xenograft model of human hepatoma and inhibition of neuronal excitability, demonstrated by blockage of voltage-sensitive tetrodotoxin-resistant (TTX-R) sodium currents in acute isolated dorsal root ganglion neurons.

CONCLUSIONS

This refolding protocol optimized for C-terminal His6-tagged scorpion rAGAP is potentially applicable to similar long-chain and cysteine-rich toxins.