菠菜43kD叶绿素a结合蛋白编码基因克隆及原核表达条件优化  

Cloning and prokaryotic expression of spinach 43 kDa chlorophyll a binding protein gene in Escherichia coli

作　　者：刘骥[1,2] 王豪[1] 肖清洁[1] 谢思思[1] 杜林方[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064 [2]西南民族大学生命科学与技术学院,成都610041

出　　处：《四川大学学报（自然科学版）》2015年第1期199-204,共6页Journal of Sichuan University(Natural Science Edition)

基　　金：国家973计划项目(2009CB118502);国家自然科学基金资助项目(30870181);教育部博士点基金(NO.20070610168)资助;西南民族大学中央高校基本科研业务费专项资金资助(11NZYQN31)

摘　　要：将菠菜43kD叶绿素结合蛋白编码基因亚克隆至原核表达载体pET-28a上,构建重组表达质粒pET-28a-psbC转化感受态E.coli BL21(DE3),经IPTG诱导实现了外源基因的可溶性表达.探讨了含组氨酸标签融合蛋白的最佳表达条件(包括表达单克隆、时间、温度对表达的影响).利用Ni2+-Sepharose 4Fast Flow亲合层析纯化出His-apo CP43蛋白.体外重组实验证实His-apo CP43能特异结合叶绿素a,经过温和电泳分离纯化所得体外重组色素蛋白复合物具有与提取自类囊体膜的天然CP43相似(但不完全相同)的荧光特性.The psbC gene isolated from Spinach was expressed in Escherichia coli in soluble state.The recombinant expression plasmid pET-28a-psbC containing spinach apo-CP43 gene was transformed into competent cell of E.coli BL21 （DE3）.The positive colony expressed effectively His-tag containing solu-ble apo-CP43 fusion protein in E.coli BL21 （DE3）when induced by IPTG under the optimal condition. The heterogeneous expression condition,including the expression monocolony,time,IPTG and temper-ature,was explored further.Near-homogeneous purified His-apo-CP43 fusion protein judged by SDS-PAGE was obtained by Ni-Sepharose Affinity Chromatography.In vitro reconstitution experiment was carried out and the formation of stable chlorophyll a-protein complex was analyzed by partially denatu-ring polyacrylamide green gel electrophoresis.After electrophoresis,the gel band containing blue-green colour was excised and measured by fluorescence emission and excitation spectra.The overall spectrum is （in a first approximation）similar to native CP43.

关 键 词：43kD叶绿素a结合蛋白 原核表达 条件优化 色素重组 荧光性质 

分 类 号：Q7[生物学—分子生物学]