The. Huwentoxin - I (of Hwtx-i) In The Pasteurized Yeast Expression

Huwentoxin- I (HWTX- I )is a neurotoxin purified from the vemon of the Chinese bird spider Selenocosmia huwena which consists of 33 amino acid residues , HWTX- I is a reversible presynaptic inhibitor which can selectively inhibit N-type Ca2+ channel. Recent research study shows that it is a peptide with analgesia activity, so it is promised to be exploited into a new kind of analgesia medicine.The synthetic HWTX- I gene was expressed in a new eukaryotic expression system-Pichia pastoris which has the advantage of high stability ,high secretion and high expression. The HWTX- I gene was chemically synthesized according to its known cDNA sequence,the gene was inserted into vector pPIC9K which contained AOXj promotor and the sequence of a secreting signal peptide- a-factor, the cloning pPIC9K/HWTX- I was constructed and confirmed by two-step PCR and DNA sequence analysis,then it was transformed into host strain GS115, A His+Muts cell line was screened and multicopy transformants were screened by various G418 concentrations, the multicopy transformant was named GH1.GH1 was cultivated in flasks. After 6 days of induction by 0.5% methanol,the supernatant was checked by 16.5% Tricine-SDS PAGE, which showed there was a band in the position of 3.5-6.1KD, then it was isolated and desalted by ultrofiltration followed by ion exchange of CM column, after reverse phase HPLC of Ci8 and vacuum drying,the purified rHWTX-1 was obtained which was proved to be correct recombinant HWTX- I by Tricine SDS-PAGE,MALDI-TOF Mass Spectrometry,amino acid composition analysis, the N-terminal amino acid sequence and its biological activity,the final field of thepurified rHWTX- I was about 80mg/L,accounting for 23.6% of it total secretory proteins.