镁螯合酶CHLI和CHLD亚基的表达纯化及其稳定复合体的鉴定

摘要

为明确植物光合作用中镁离子螯合酶CHLI与CHLD亚基形成复合物CHLI-CHLD这一重要过程的机制,首先需要获得可溶性CHLI和CHLD蛋白并确定其复合体装配。通过分子克隆技术构建截断的CHLI和CHLD重组体表达质粒。将其分别转化至大肠杆菌BL21(DE3)中,添加异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达。通过Ni琼脂糖树脂(Ni sepharose 6 Fast Flow)和谷胱甘肽琼脂糖树脂(Glutathionsepharose 4 Fast Flow)亲和层析纯化可溶性重组蛋白。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和TEV酶切试验验证目标蛋白。采用GST-Pulldown试验鉴定CHLI与CHLD的相互作用。通过凝胶过滤层析和SDS-PAGE测定复合体中CHLI与CHLD蛋白的比例。结果表明：成功构建了截断的CHLI和CHLD重组体表达质粒pET-28a-CHLI (13-357),pET-28a-CHLD (20-676)及pGEX-4T-2-CHLD (20-767)；在18℃,0.2mmol•L-1 IPTG条件下诱导16h后,成功获得了可溶性的CHLI (13-357),CHLD (20-676)和GST-CHLD (20-676)重组表达蛋白；CHLI与CHLD存在直接相互作用且需要Mg2+和ATP；稳定复合体CHLI-CHLD的亚基比例为1︰1。综上表明原核表达及纯化镁离子螯合酶CHLI与CHLD亚基蛋白,在Mg2+和ATP存在的条件下通过凝胶过滤层析可获得1︰1的稳定复合体CHLI-CHLD。%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.