Functional Expression Of Spider Neurotoxic Peptide Hainantoxin-Ⅳ In E.Coli

Hainantoxin-IV is a kind of neurotoxin extracted from selenocosmia hainana which is found in resent years. It contains 35 amino-acids. Its sequence is NH2-ECLGFGKGCNPSNDQCCKSSNLVCSRKHRWCK YEI-COOH, forming firm ICK motif with three pairs of disulfide bonds. The toxin is a kind of TTX-sensitive Na+ channel inhibitor.Using pET expression system, recombinant HNTX-IV (rHNTX-IV) toxin was produced successfully. According to coding sequence, primers of HNTX-IV was designed. Using PCR to amplify HNTX-IV fragment, it was inserted into pET40 vector directionally. The construct was transformed into BL21 (DE3) expression strain. To induce exogenous protein expression, we adopted two different ways:IPTG induction and auto-induction. It was made a comparison between the two ways metioned above. IPTG is lactose-like sbustance, can efficiently initiate lactose operon. IPTG induction has become one of the most widely used method in procaryotic expression. However, due to its amazing inducing ability, the expressed protain usurally exsist in inclusion body, which is hardly renaturated. Because thoxin peptide contains several disulfide bonds, it’s even more complicated to obain toxin whit natural activity. The expressed fusion protein with DSB tag secretes into periplasmic space of E.coli can be separated through osmotic shock. After purified by Ni column affinity chromatograph and digested by enterokinase, peptide was isolated. Further purification was developed by RP-HPLC and pure rHNTX-IV was obtained. The rHNTX-IV was analysised by SDS-PAGE electrophoresis and mass spectrogram. The molecular mass of rHNTX-IV is 3985.75 Da, identical to natural toxin. The rHNTX-IV displays the same biological activity to natural toxin by patch clamp assays.