Mediation Fu (g.i Ntermedius) Snake Venom Serine Protease Gi - Tle1 Yeast Expression

G.intermedius subordinates to the Genus Gloydius, the subfamily Crotalinae of Viperidae. It is the most toxic one among six pit-vipers. G.intermedius is widely distributed in Northwest China (including Gansu, Qinghai, Xinjiang, Ningxia and the north of Shaanxi province) and some areas of North China (Inner Mon golia and the Loess Plateau area in northern of Shanxi province), and is scattered in republic of Mongolia and the southern area of Siberia. Its venom has important application value in basic scientific research and medical pharmacology study. In the past, the expression of snake venom proteins in the prokaryotic cells had been explored, but the products of prokaryotic expression formed inclusion body usually and the renaturation were difficult, which was not good to the further purification of the product and study of activity. In view of this, we tried to obtain the snake venom serine proteinases (SVSPs) in the yeast expression system.Thrombin-like enzyme1(GI-TLE1) gene, which came from venom gland cDNA library of G. intermedius, has been amplified by PCR. The amplified product was sub-cloned into the yeast expression vector pPICZaA, after screening by PCR and verifying by DNA sequencing, the recombinant yeast expression vector has been successfully constructed. Then the recombinant plasmid was linearized and electroporated into Pichia pastoris GS115and KM71respectively. At the same time, We used YPDZ screened plate which contained different concentrations of Zeocin, followed by100μg/mL,200μg/mL and300μg/mL to get the positive clones of high copy number, and the integer of GS115positive clones of high copy number was identified, the results showed that the GS115transformants was Mut+phenotype. At last, the genomic DNA of the transformants was extracted. After identifying by PCR, the target gene has been successfully integrated into GS115and KM71yeast genome. At so the induction and expression stage, we selected28℃,250rpm/min,0.5%methanol to induce the expression of exogenous protein. At the same time, we optimized the time condition and took samples at different time pionts (Oh,12h,24h,36h,48h,60h,72h,84h,96h) respectively, and then we added0.5%methanol into the medium every24h in order to maintain continuous induction. SDS-PAGE analysis showed that GI-TLEl had been successfully expressed in GS115transformants at36h and48h, but had not been expressed at any time point in KM71. Then we optimized the methanol condition and induced at0.5%,1%and1.5%respectively, the results showed that the total protein content in the supernatant of GS115and KM71was the highest when induced with0.5%methanol. Finally, the hydrolase activity of the expressed GI-TLE1was identified with BApNA as substrate. However, the expression level was too low, the color change of the reaction solution was not obvious. So we can not evaluate preliminarily whether the expressed protein has the biological activity. The strength of the activity will be identified quantitatively after improving production and purifying by Ni2+-IDA resin affinity column. Even so, our present work will undoubtedly pave the way for the expression of snake venom proteins in Pichia pastoris.