平颏海蛇碱性磷脂酶A_2的融合表达、纯化及活性特征

 将编码平颏海蛇碱性磷脂酶A2 的基因 (PLA2 9)克隆于硫氧还蛋白基因融合表达载体pPETTRX的trxA基因的 3′末端 ,构建符合读码框的融合基因 .2 5℃下经IPTG诱导 ,该融合蛋白在大肠杆菌中以可溶形式表达 ,表达量达 2 0 %以上 .利用金属螯合亲和层析和凝胶过滤层析两步纯化 ,得到纯度 85%的融合蛋白 .经肠激酶切割和离子交换柱层析进一步纯化后 ,得到浓度 95%以上的成熟PLA2 9.对重组PLA2 9进行了Western印迹检测 .重组PLA2 9具有与天然PLA2 相近的酶活性 ;并具有对HL60等几种肿瘤细胞的细胞毒作用 ,这在海蛇PLA2 中是首次报道 .平颏海蛇碱性PLA2 融合表达及快速纯化系统的建立 ,为深入开展其结构与功能关系研究奠定了基础

Fusion Expression,Purification and Activity Characterization of Alkaline Phospholipase A_2 from Lapemis hardwickii Gray

Phospholipase A 2(PLA 2) are the major components of snake venoms.The gene encoding alkaline PLA 2(PLA 2 9) from Lapemis hardwickii Gray was cloned into the 3′ end of the thioredoxin gene ( trxA ) in plasmid pETTRX to construct the pETRX PLA 2 9 fusion expression vector.The TRX PLA 2 9 was expressed as a soluble protein in E.coli induced by IPTG at 25℃,and amounted to more than 20% of the total bacterial proteins.A fusion protein of PLA 2 9 with over 85% purity was obtained through two step purification consisting of immobilized metal chelate affinity chromatography and gel filtration.After cutting the fusion protein with enterokinase and further purification by cation exchange chromatography on SP Sepharose fast flow,the mature PLA 2 9 with 95% purity was obtained.The recombinant PLA 2 9 was detected by Western blot analysis.The PLA 2 9 has the similar enzymatic activity to the natural PLA 2 and has the cytotoxic activity on several tumor cell lines such as promyelocytic leukemia HL60.This is the first report on the sea snake PLA 2 research.The successful expression and rapid purification of recombinant PLA 2 9 would facilitate the further study of structure function relationship of PLA 2 9 and the potential medicine development.