利用大肠杆菌高效表达人载脂蛋白AI及其性质分析  

Over-expression in Escherichia coli and Characterization of Apolipoprotein AI

作　　者：丁满生[1] 马文峰[1] 张梅芳 刘大涛 郭美锦[1] 庄英萍[1] 储炬[1] 张嗣良[1] 龚邦强 

机构地区：[1]华东理工大学生物反应器工程国家重点实验室,上海200237 [2]上海凯曼生物科技有限公司,上海200233 [3]上海信谊药业有限公司药研所生物制药研究室,上海200085

出　　处：《生物工程学报》2005年第2期198-203,共6页Chinese Journal of Biotechnology

基　　金：国家高技术研究发展计划 (863计划 )项目 (No.2 0 0 2AA2 170 2 1);国家自然科学基金重大科技专项 (No.2 0 0 2AA2Z3 45 1)资助~~

摘　　要：载脂蛋白AI(apolipoproteinAI,apoAI)是高密度脂蛋白HDL的主要组成成分 ,流行病学研究表明apoAI的含量决定血浆中HDL的高低 ,而HDL具有胆固醇逆向转运的功能 ,起到降低冠心病发生概率的作用。通过大肠杆菌表达系统来生产apoAI的蛋白原proapoAI,蛋白的表达形式为包涵体 ,通过疏水柱进行柱上复性。同时在proapoAI和apoAI之间设计一个酸水解位点 ,通过将蛋白原proapoAI进行酸水解来得到成熟蛋白apoAI。最终得到的成熟蛋白apoAI在结构分析和脂结合特性上都与天然的apoAI相似 ,从而为该蛋白的工业生产上提供了一个良好的基础。Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E.coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E.coli including a plasmid of P RP L tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and l

关 键 词：载脂蛋白AI 包涵体 疏水柱 复性 

分 类 号：R346[医药卫生—基础医学]