Gene Cloning,Prokaryotic Expression And Purification Of Toxins From Scorpion Lychas Mucronatus

Lychas mucronatus is one of the scorpion species distributed widely in southern China and Southeast Asia.Studies have shown that Lychas mucronatus venom contains a large number of biological active ingredients,and it is a treasure trove of natural resources to develop many new drugs and therapeutic agents.Lychas mucronatus venom is rich in many kinds of small molecule toxins containing three or four pairs of disulfide bonds.These peptide toxins can interact specifically with a variety of voltage-sensitive channels,changing their dynamic characteristics or blocking the channel ion currents.Hence these scorpion toxins have extremely important physiological functions and research value.However,separating scorpion toxins directly from scorpion venoms is quite difficult,and the presence of disulfide bonds also makes it difficult to synthesize peptide toxins in chemical methods.Consequently,genetic engineering technologies have been used to obtain high yield of active peptide toxins.GT028649 and GT028758 are toxins obtained from the dbEST database,which possibly have similar biological activities with natural scorpion toxins.They are composed of 62 and 63 amino acids respectively,containing eight cysteines and four pairs of disulfide bonds.The DNA sequences of these two toxin molecules were inserted into the expression vector pET-His-SUMO by RF cloning,and the recombinant protein was expressed in E.coli Shuffle strain using E.coli expression system.The expressed fusion protein was purified by nickel column affinity chromatography and ultrafiltration.The His-SUMO fusion tag was separated from the target protein by Ulp1 enzyme digesting.The target protein was further purified by reversed-phase high performance liquid chromatography(RP-HPLC)to improve the purity.The results showed that the molecular weight of the two proteins were 7279.3230 Da and 7280.6279 Da respectively,which are consistent to their theoretical molecular weight(7278 Da and 7281 Da respectively).This indicates that the toxin peptides containing four pairs of disulfide bonds were successfully expressed and purified.In the subsequent experiments,the electrophysiological activity of these two protein products will also be detected.This study laid the foundation for the further exploration on the structure and function of scorpion venom as well as the development of new peptide drugs.