菠菜43 kD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(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)；王豪(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)；肖清洁(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)；谢思思(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)；杜林方(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)
Author：LIU Ji(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；WANG Hao(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；XIAO Qing-Jie(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；XIE Si-Si(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)；DU Lin-Fang(Key laboratory of Bio resources and Eco environment of Ministry of Education,College of Life Sciences, Sichuan University)
收稿日期：2014-04-15 年卷（期）页码：2015,52(1):199-204
期刊名称：四川大学学报: 自然科学版
Journal Name：Journal of Sichuan University (Natural Science Edition)
关键字：43 kD叶绿素a结合蛋白; 原核表达; 条件优化; 色素重组; 荧光性质
Key words：His apo CP43 fusion protein, prokaryotic expression, purification, reconstitution experiment, fluorescence
基金项目：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)
中文摘要
将菠菜43 kD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 4 Fast 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.
英文摘要
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 soluble 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 temperature, 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 denaturing 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.
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