一种缺失核定位信号区的hbFGF的表达及纯化(英文)  

Expression and Purification of a hbFGF Lacking Nuclear Localization Signal

作　　者：吴晓萍[1] 苏志坚[1] 郑青[1] 吴思娴[2] 冯雅[2] 曲红艳[2] 许华[1] 李校堃[1] 

机构地区：[1]暨南大学药学院,广州510632 [2]暨南大学医药生物技术研究开发中心,广州510632

出　　处：《中国药科大学学报》2005年第3期272-275,共4页Journal of China Pharmaceutical University

基　　金：国家高技术研究发展计划("八六三"计划)资助项目(No.2001AA215131,2002AA2Z3318);广东省自然科学基金资助项目(No.010424)~~

摘　　要：目的:为了研究缺失核定位信号区的人碱性成纤维细胞生长因子(hbFGF)独特的转运机制,在大肠杆菌BL21(DE3)中高效表达和纯化了缺失核定位信号区的hbFGF。方法:将PCR扩增得到的hbFGFcDNA片断克隆到表达载体pET3c中,重组质粒转化大肠杆菌BL21(DE3),IPTG诱导表达,通过离子交换和肝素亲和层析从菌体上清中纯化目标蛋白,MTT法检测促分裂活性。结果:hbFGF的表达量约为全菌体蛋白的20%,纯化的缺失核定位信号区的hbFGF的促分裂活性与缺失核定位信号区的hbFGF标准品相当。结论:BL21(DE3)/pET3c表达系统能高效表达缺失核定位信号区的hbFGF,纯化得到的hbFGF可用于进一步的研究。AIM:To study the mechanism of the unique export of one of human basic fibroblast growth factor (hbFGF) forms lacking the N-terminal nuclear localization signal (NLS),we high expressed and purified this hbFGF form in E.coli strain BL21(DE3).METHODS:The cDNA fragment of the hbFGF amplified by polymerase chain reaction (PCR) was cloned into the expression vector pET3c and expressed in BL21(DE3) by IPTG induction.The expressed hbFGF was purified by ionic exchange and heparin affinity chromatography from the supernatant of bacteria lysate.The mitogenic activity was measured by MTT.RESULTS:The expression level of hbFGF in E.coli was about 20% of the total cellular protein.The appreciable mitogenic activity of the purified hbFGF was comparable to that of hbFGF standard.CONCLUSION:The BL21(DE3)/ pET3c expression system could be used to high express hbFGF lacking NLS.The purified recombinant hbFGF was prepared and sufficient for further study.

关 键 词：人碱性成纤维细胞生长因子 核定位信号 纯化 

分 类 号：Q786[生物学—分子生物学]