重组人载脂蛋白AI米兰突变体在大肠杆菌中的可溶性表达

载脂蛋白AI米兰突变体(apoliproteinA-I-Milano ,AIM)是载脂蛋白AI的天然突变体,具有比载脂蛋白AI更强的抗动脉粥样硬化症的作用。将AIM基因N_末端的氨基酸密码子优化后,克隆至表达载体pET2 2b ,重组质粒转化BL2 1(DE3)宿主菌,用IPTG诱导表达。AIM以可溶形式表达,约占菌体总蛋白的38%。表达产物经ButylSepharose 4F .F疏水层析和QSepharoseH .P .阴离子交换层析后,再用Vivaspin2 0 (30 0 0 0MW)进行超滤,AIM单体的纯度达95 %以上。AIM单体与脂结合活性表明,AIM单体结合脂的速度比apoA_I慢,但结合脂的量比apoA_I高。这项研究为AIM的结构和功能,特别是临床应用研究奠定了基础。

Soluble Expression of Recombinant Human Apoliprotein A-I-Milano in Escherichia coli

Apolipoprotein A-I-Milano(AIM),a natural variant, not only inhibits the initiation and progression of atherosclerosis,but also makes the preexisting atherosclerotic lesions regress.AIM gene, at which N-terminal codens were optimized,was subcloned into the expression vector of pET22b.Recombiant plasmids were transformed into E.coli strain BL21(DE3) and induced with IPTG.The expressed apoliprotein A-I-Milano was soluble in E.coli and was about 38% of total cell lysate.Purified by Butyl Sepharose 4F.F hydrophobic chromatography and Q Sepharose H.P. anion exchange chromatography,followed by ultrafiltration with Vivaspin 20(30 000MW),AIM monomer was obtained in a purity of more than 95%. Activity assay of binding of AIM monomer to lipid indicates that association of AIM monomer with DMPC is slower than normal apoA-I but DMPC number associated by AIM monomer is more than by apoA-I.This results will be important for studying structure,function of AIM,specially clinical application.