脂多糖应激新分子融合蛋白的高表达及纯化  

High efficiency expression and purification of lipopolysaccharide respond gene fusion protein

作　　者：杜可军[1] 常文辉 侯立朝[3] 骆文静[1] 刘承利[4] 陈苏民[4] 陈景元[1] 柴玉波[4] 

机构地区：[1]第四军医大学劳动与环境卫生学教研室,陕西西安710032 [2]陕西省疾控中心 [3]第四军医大学西京医院麻醉科 [4]第四军医大学生物化学与分子生物学教研室

出　　处：《毒理学杂志》2006年第5期291-293,共3页Journal of Toxicology

基　　金：国家自然科学基金资助项目(30170361;30400413;30571586);第四军医大学博士后基金资助项目(00001149)

摘　　要：目的在原核表达系统中表达脂多糖应激新分子编码的蛋白质。方法PCR扩增HlrgcDNA编码区,克隆入表达载体pcTAT中,构建成融合6His的表达载体pcTAT-lrg,转化E.coli BL21(DE3),以IPTG诱导。表达产物用SDS-PAGE及凝胶光密度扫描分析,用Ni-NTA亲和层析柱纯化。结果经过IPTG诱导4 h,表达出相对分子质量(Mr)约25 000的蛋白,占菌体总蛋白的51%。表达产物为可溶性的,用Ni-NTA亲和层析柱纯化的表达蛋白达到电泳纯。加入培养液中的纯化蛋白,可在30 min内进入HEK293细胞。结论在E.coli中成功地高表达人Lrg融合蛋白,并对其进行初步纯化,为人Lrg功能的研究打下基础。Objective To expression the recombinant human lipopolysaccharide respond gene （Lrg） protein in E. coli. Methods The encoding region of human Lrg cDNA was amplified by PCR, then subcloned into the prokaryotic expression vector pcTAT to construct pcTAT-hLrg. 6His-TAT was fused with coding sequence of hLrg cDNA in-frame with E. coli BI21 （ DE3）. Results 6His- TAT-hLrg had been expressed under IPTG induction, the expressed product accounted for 51% of the total protein, and was soluble. The 6His-TAT-hLrg purified through Ni-NTA affinity chromatography column reached electrophoretical pure. The purified protein could be found in the cytoplasm of HEK293 cells within 30 minutes. Conclusion Human Lrg fusion protein was expressed in E. coli successfully and high-efficiency, and Lrg was preliminary purified through Ni-NTA column, Which laid the foundation for further study of the function of human Lrg,

关 键 词：脂多糖应激新分子 蛋白融合表达 克隆 纯化 

分 类 号：Q271[生物学—细胞生物学]