人可溶性APO2L/TRAIL基因的克隆及其在大肠杆菌中的表达

Molecular Cloning and Expression of Human Soluble APO2L/TRAIL Gene in Escherichia coli

目的 利用基因工程技术原核表达人可溶性APO2L并复性纯化成具生物活性形式。方法 用PCR方法扩增APO2LcDNA(密码子 1 1 4～ 2 81 )并克隆至表达载体 ,重组体转化大肠杆菌JF1 1 2 5摇瓶发酵 ,筛选高表达克隆于发酵罐经温度诱导表达。经盐酸胍溶解 ,空气氧化法稀释复性 ,超滤 ,Q SepharoseFF阴离子交换柱层析等方法纯化人可溶性APO2L。利用流式细胞术检测产物对细胞的诱导凋亡活性。结果 表达产物约占菌体总蛋白质的 2 0 %并在菌体内形成了包涵体 ,SDS PAGE鉴定表达产物相对分子质量约 2 1 0 0 0。经上述纯化蛋白质纯度达 90 %以上 ,蛋白质可溶性好。观察到人可溶性APO2L诱导人宫颈癌HeLa细胞发生明显的凋亡。

Purpose To express soluble and functional human APO2L in Escherichia coli and refold it into a functional form. Methods Human APO2L condons 114-281 were amplified by polymerase chain reaction and subcloned into expression vector.The product was transfected into E.coli JF1125 and expressed by temperature inducement in shaking bottle.The selected high expression clone was expressed in bioreactor.Soluble human APO2L was obtained by a protocol,which includes dissolving by Gu·HCl,refolding by Air Oxidation,diafiltration and Q?Sepharose ino exchange chromatography. Results SDS?PAGE analysis revealed that expression product was Mr.2.1,about 20% of total bacteria protein and formed inclusion body.The purity of soluble human APO2L was more than 90%.Induction of Apoptosis by the product became conscious in Hela Cells. Conclusions The product expressed in Escherichia coli has similar activity to that generated in cells.