具有生物活性的人带3蛋白胞质片段融合蛋白的克隆、表达与纯化  

Cloning, Expression and Purification of a Functional Fusion Protein with Cytoplasmic Domain of Human Band 3

作　　者：郑玉娟[1] 邢菲菲[1] 孙立伟[1] 刘立岩[1] 赵大庆[1] 倪嘉缵[1] 

机构地区：[1]中国科学院长春应用化学研究所

出　　处：《高等学校化学学报》2003年第3期386-389,共4页Chemical Journal of Chinese Universities

基　　金：国家自然科学基金重大项目 (批准号 :2 9890 2 80 )资助

摘　　要：用 PCR法从质粒 p HB3中扩增了人红细胞带 3蛋白胞质片段 (CDB3 )基因 .PCR产物经限制性内切酶切割后与多克隆位点处带有编码 6个组氨酸序列的高效表达载体 p ET2 8b连接 ,构建为重组子p CDBHistag.重组子经酶切及序列测定后在大肠杆菌 BL2 1 (DE3 )中获得高效表达 ,可溶性目的蛋白占菌体总蛋白的 4 0 %左右 .C端带有 6个连续组氨酸的带 3蛋白胞质片段作为融合蛋白不仅可以降低宿主菌蛋白酶对其水解程度 ,而且简化了目的蛋白的纯化过程 .经一步螯合 Ni2 +的亲和层析获得了电泳纯的带 3蛋白胞质片段融合蛋白 .活性测定结果表明 ,带 3蛋白胞质片段融合蛋白能够抑制醛缩酶 (Aldolase)活性的70 % ,与文献报道的人红细胞内带 3蛋白胞质片段具有相同的功能 .The cytoplasmic domain of human erythrocyte band 3(CDB3) gene was amplified by standard PCR method from plasmid pHB3 and constructed to be a recombinant plasmid pCDBHistag with an expression vector pET28b. Recombinant CDB3 fusion protein with Polyhistidine-tag was strongly expressed as soluble protein in E coli BL21(DE3) after induction with isopropyl-β-D-thiogalactopyranoside. Affinity chromatograpy chelating with Ni 2+ was employed to purify the recombinant fusion CDB3 protein. About 70% of aldolase activity could be inhibited by purified recombinant CDB3 similar as the result in human erythrocyte. It suggested that aldolase could bind with the purified CDB3 fusion protein in vitro. The high-level expression and easy purification of this recombinant protein should permit thorough studies of the interaction mechanism of CDB3 with its peripheral binding proteins.

关 键 词：带3蛋白胞质片段 融合蛋白 纯化 亲和层析 基因表达 基因克隆 

分 类 号：Q51[生物学—生物化学] Q78