Impact Of Disulfide Bridges In Scorpion Active Peptide AS To The Analgesic Activity

The activity peptide from Buthus martensii Karsch (BmK AS) is a kind of scorpion venom, which has the activity of analgesia and antimicrobia. The full length of the sequence of BmKAS is 198bp, which encode 66 amino acid residues. It’s known that multiple disulfide bonds cross-link the polypeptide chain to provide most of their stabilizing energy. Therefore, the disulfide bridges are crucial for the scaffold of scorpion toxins. Now it is not reported the relation of disulfide bonds and the analgesic activity of BmK AS. Here we use mutagenesis analysis to research the two disulfide bonds in BmK AS.BmK AS was added a His Tag to combinate rBmK AS to abtain fusion protein and purify. The rBmK AS maintained a similar analgesic activity to the natural. Cysteines of rBmK AS were replaced by serines to research the impact of disulfide bridges to the analgesic activity.Different AS genes were obtained by PCR using different primers from pSYPU/AS. Cysteines at 12,62,12 and 62,16 were replaced by serines to abtain four mutant frags by PCR. Cysteine at 37 was replaced by serine to abtain one mutant frag by two-step PCR. The five mutants were cloned to pSYPU-lb to combinate pSYPU/AS C12S, pSYPU/AS C62S, pSYPU/AS C12-62S, pSYPU/AS C16S and pSYPU/AS C37S. Transformed them into the E. coli BL21(DE3), and then induced the expression vector by adding IPTG. The five proteins were soluble.Then the five target proteins were purified from the supernatant of E. coli broth by metal chelating affinity chromatography. And the analgesic activity of them was detected by an animal model of twisting action of the mice induced by acetic acid. Pharmaceutical tests showed that the recombinant AS has the analgesic activity on mice. When giving same dosage (0.1364μmol/kg) of the different proteins (rBmK AS C12S, rBmK AS C62S, rBmK AS C12-62S, rBmK AS C16S and rBmK AS C37S), the rates of inhibition of twisting action of the mice were 42.1%,45.6%,38.5%,41.2%and 43.6%respectively. The relationship between disulfide bridges and analgesic activity of AS was further explored by studying of the activity of those five mutants of BmK AS.