四膜虫金属硫蛋白MTT2在大肠杆菌中的表达与纯化  

Expression and Purification of Tetrahymena elliotti MTT2 in Escherichia coli

作　　者：郭荣[1] 许静[1] 王伟[1] 

机构地区：[1]山西大学生物技术研究所化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《山西大学学报（自然科学版）》2016年第2期301-306,共6页Journal of Shanxi University(Natural Science Edition)

基　　金：国家自然科学基金(31471999;315672253);山西省国际合作计划项目(2015081032);山西省自然科学基金(2015011178)

摘　　要：金属硫蛋白是一种在多种生物中存在的多功能蛋白,纤毛虫金属硫蛋白在结构和功能上具有物种特殊性。从四膜虫Tetrahymena elliotti中鉴定到一种新的金属硫蛋白基因Te-MTT2,该基因全长273bp,无内含子,编码90个氨基酸。为了分析Te-MTT2的功能,分别构建了重组质粒pGST-Te-MTT2和pSUMO-Te-MTT2并转化大肠杆菌BL21.0.05mmol/L IPTG诱导大肠杆菌BL21/pGST-Te-MTT2和BL21/pSUMO-Te-MTT2表达融合蛋白GST-Te-MTT2和SUMO-Te-MTT2.GST-Te-MTT2的表达量占到总蛋白的16.7%,其中29.9%可溶;SUMOTe-MTT2的表达量占到总蛋白的14.2%,其中76.2%可溶。SUMO-Te-MTT2经Ni柱亲和纯化获得电泳纯的蛋白。85℃,15min温浴后,SUMO-Te-MTT2蛋白在10mmol/L PBS溶液中稳定存在。结果表明SUMO表达系统提高了Te-MTT2的可溶性表达,Te-MTT2的表达纯化为进一步分析其生物学功能奠定了基础。Metallothioneins（MTs）are multifunctional proteins,which have been found widely distributed in different organisms.Ciliate MTs have longer sequence features and special function.A novel MT gene Te-MTT2 was identified fromTetrahymena elliotti.Te-MTT2 is 273bp and encodes 90 amino acids.To analyze the function of Te-MTT2,recombinant plasmids pGST-Te-MTT2 and pSUMO-Te-MTT2 were constructed and transformed into E.coli BL21,respectively.GST-Te-MTT2 and SUMO-Te-MTT2 were expressed fromE.coli BL21/pGST-Te-MTT2 and E.coli BL21/pSUMO-Te-MTT2 by 0.05mmol/L IPTG induction,respectively.The expression level of GST-Te-MTT2 reached 16.7% of the total protein,of which the soluble GST-Te-MTT2 was up to 29.9%;the expression level of SUMO-Te-MTT2reached14.2% of the total protein,of which the soluble SUMO-Te-MTT2 was up to 76.2%.Then,SUMO-TeMTT2 was purified by Ni affinity chromatography.SUMO-Te-MTT2 was stable at 85℃for 15 minutes.The results showed that SUMO tag is effective for Te-MTT2 soluble expression.The recombinant expression of Te-MTT2 is important for its functional research and structure analysis.

关 键 词：金属硫蛋白 原核表达 纯化 四膜虫 

分 类 号：Q786[生物学—分子生物学]