沈阳农业大学学报2016，Vol.47Issue(3)：283-290,8.DOI:10.3969/j.issn.1000-1700.2016.03.005
镁螯合酶CHLI和CHLD亚基的表达纯化及其稳定复合体的鉴定
Expression and Purification of CHLI and CHLD, Identification of Their Stable Complex
摘要
Abstract
In order to determine the important mechanism that CHLI and CHLD subunits of Mg2+-chelatase in photosynthesis form the complex CHLI-CHLD, the soluble CHLI and CHLD proteins in vitro are required to be prepared and assembled. The truncated CHLI and CHLD were constructed into the prokaryotic expression vectors through molecular cloning techniques. Each of them was transformed into Escherichia coli BL21(DE3), and induced to express the target proteins by addition of isopropylthio-β-d-galactoside (IPTG). The recombinant target proteins were purified by the affinity chromatography column filled with Ni sepharose 6 Fast Flow or Glutathionsepharose 4 Fast Flow. The soluble recombinant proteins were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and TEV protease digestion assays. The GST-Pulldown assays testified that a direct interaction between CHLI and CHLD. The stoichiometry of CHLI and CHLD in complex was measured by gel filtration chromatography and SDS-PAGE. Our results showed that the truncated CHLI and CHLD were successfully constructed into the prokaryotic expression vectors to form recombinants including pET-28a-CHLI (13-357), pET-28a-CHLD (20-676) and pGEX-4T-2-CHLD (20-767); the soluble recombinant proteins CHLI (13-357), CHLD (20-676) and GST-CHLD (20-676) were successfully gained after induction by 0.2 mmol•L-1 IPTG for 16 h at 18℃; a direct interaction between CHLI and CHLD existed in the presence of Mg2+ and ATP; the stable complex of CHLI-CHLD with 1:1 stoichiometry was obtained. Together, Mg2+-chelatase subunits CHLI and CHLD proteins were obtained through prokaryotic expression and purification, and the stable complex of CHLI-CHLD with 1︰1 stoichiometry in the presence of Mg2+ and ATP was acquired by the gel filtration chromatography.关键词
CHLI/CHLD/原核表达及纯化/蛋白复合物Key words
CHLI/CHLD/prokaryotic expression and purification/protein complex分类
农业科技引用本文复制引用
张星亮,彭熹,邓翔,刘扬,崔香淑..镁螯合酶CHLI和CHLD亚基的表达纯化及其稳定复合体的鉴定[J].沈阳农业大学学报,2016,47(3):283-290,8.基金项目
国家自然科学青年基金项目(31300602) （31300602）
广东省自然科学基金项目(S2013040012902) （S2013040012902）
吉林省卫生厅基金项目(2013082) （2013082）
