mtPDI外源基因表达体系的构建及耐热性  

Heterologous Expression and Purification of mtPDI from Methanothermobacter thermautotrophicus in Escherichia coli

作　　者：丁霞[1,2] 闵航[3] 杨卫军[3] 

机构地区：[1]南昌大学生命科学与食品工程学院 [2]南昌大学生命科学研究院,江西南昌330031 [3]浙江大学生命科学学院,浙江杭州310058

出　　处：《南昌大学学报（理科版）》2010年第5期498-502,共5页Journal of Nanchang University(Natural Science)

基　　金：江西省自然科学基金资助项目(2009GQN0091);江西省教育厅基金资助项目(GJJ10048)

摘　　要：二硫键异构酶(PDI)是1种重要的蛋白折叠酶,它催化蛋白二硫键的形成和错误配对二硫键的重排,并有抑制错误折叠蛋白聚集的分子伴侣活性。为了研究嗜热自养甲烷杆菌二硫键异构酶(mtPDI)的功能,克隆了mtP-DI基因的ORF区,构建了融合基因原核表达载体pET-28a(+)-mtPDI。将质粒pET-28a(+)-mtPDI和pSJS1240共转化E.coliBL21(DE3)构建E.coli(pSJS1240,pET-28a-mtPDI)过表达系统,实现了融合蛋白的高效表达。另外,进行了mtPDI对大肠杆菌的热耐受性研究,E.coli(pET-28a-mtPDI)在30℃和IPTG诱导30 min后,在51℃热刺激60 min后能够提高E.coli的生存率。这一结果表明mtPDI在热损伤保护中具有关键性作用。上述实验结果为嗜热自养甲烷杆菌的二硫键异构酶的抗胁迫机制的深入研究奠定了基础。mtPDI is a protein disulfide isomerase characterized with 151 amino acid residues and a CPAC active-site from the anaerobic archaea Methanothermobacter thermautotrophicus.The potential functions of mtPDI are not clear.The potential functions of mtPDI are not clear.In the present study,the survival of mtPDI expressing Escherichia coli cells was markedly higher than that of control cells in response to heat shock(51.0 ℃).The result indicated that mtPDI plays an important role in the resistance of stress.In order to perform the assay of enzyme activities in vitro,a prokaryotic expression vector pET-28a(+)-mtPDI was constructed.The plasmids pET-28a(+)-mtPDI and pSJS1240 were transformed into Escherichia coli BL21(DE3).The fusion protein was expressed successfully in the form of inclusion body.The recombination protein of mtPDI was highly purified by chromatography of Ni-NTA Agarose,which was identifited by SDS-PAGE and HPLC.These studies may provide help to the understanding of the function of protein disulfide isomerase in archaea.

关 键 词：古菌 二硫键异构酶 嗜热自养甲烷杆菌 纯化 热耐受性 

分 类 号：Q343[生物学—遗传学]