平颏海蛇PLA_2在大肠杆菌中的融合表达

Fusion Expression of PLA_2 Gene From Lapemis hardwickii in E.coli

将编码平颏海蛇磷脂酶A2 的基因 (PLA2 9)分别克隆于硫氧环蛋白基因融合表达载体pThioHisC和pTRX的HP trxA和trxA基因的 3′末端 ,构建符合读码框的融合表达载体pThioHisC PLA2 和pTRX PLA2 。 2 5℃下经IPTG诱导 ,PLA2 融合蛋白在两种原核系统中均能获得较高表达 ,但PLA2 在pTRX系统下的表达量和蛋白溶解性优于pThioHisC系统。将两种系统下的表达产物进行金属螯合亲和层析纯化 ,Trx PLA2 融合蛋白的纯度可达 85 %以上 ,而HP Trx PLA2 不表现出对介质的亲和性 ,难以得到纯化。由此 ,将pTRX PLA2 载体系统确立为进一步大量表达和纯化的载体系统。

The gene encoding PLA\-2(PLA\-2\|9) from Lapemis hardwickii Gray venom was cloned to the 3'and of the thioredoxin gene (HP\|trxA and trxA)in plasmid pthioHisC and pTRX to construct the pThioHisC\| PLA\-2 and pTRX\| PLA\-2 fusion expression vector.The fusion protein of PLA\-2 can be expressed in the two different systems induced by IPTG at 25℃,but the expression level and the solubility of the fusion protein in pTRX were better than that in pThioHisC.The expressed product in the two systems were purified by immobilized metal\|chelate affinity chromatography.The Trx\|PLA\-2 fusion protein with over 85% purity was obtained and HP\|Trx\|PLA\-2 fusion protein can not be purified since it dose not exhibit affinity to the medium.So,the pTRX\| PLA\-2 vector system was established for the large\|scale expression and purification.