Purification of a minimal complex for oxygen evolution from the recombinant cyanobacteria that carry thermostable photosystem II

光系统II反应中心亚单位，d1-d2杂二聚体催化分解水和放氧反应。为了建立一种分离异源二聚体的方法，通过rps12介导的基因置换构建长链球菌异二聚体重组载体，在嗜热蓝细菌中表达了外生型耐热链球菌D1D2蛋白。将携带rps12R-43突变的GRPS1转化为GRPS201和GRPS220菌株。GRPS201携带替换的psbA1基因，而GRPS220同时携带替换的psbA1和psbD1基因。从这些蓝藻中制备类囊体膜蛋白，并使用针对T.viscanusD1-1蛋白的抗体进行蛋白质印迹分析。虽然在GRPS201和GRPS220菌株中检测到了D1蛋白，但其大小比硫杆菌天然的D1-1大2 kDa，表明其C端16个氨基酸没有被加工。另一方面，还纯化了硫杆菌HSPA分子伴侣蛋白，为去除类囊体膜上的聚集蛋白，获得纯化的D1-D2杂二聚体蛋白奠定了基础。

Photosystem II reaction center subunits, D1-D2 heterodimer catalyze water splitting and oxygen evolving reaction. To establish a method to isolate the heterodimer, D1 and D2 proteins from a thermophilic cyanobacterium Thermasynechococcus vulcanus were expressed in a mesophilic Synechococcus elongatus by constructing the recombinant S.elongatus via rps12-mediated gene replacement. A GRPS1 carrying an rps12R-43 mutation was transformed to obtain GRPS201 and GRPS220 strains. GRPS201 carries a replaced psbA1 gene, whereas GRPS220 carries both replaced psbA1 and psbD1 genes from T.vulcanus Thylakoid membrane proteins were prepared from these cyanobacteria, and analyzed by Western blotting using an antibody specific for T.vulcanusD1-1 protein. Although D1 protein was detected in GRPS201 and GRPS220 strains, the size of D1-1 was larger by 2kDa than the native D1-1 from T.vulcanus, suggesting that C-terminal 16 amino acids were not processed. On the other hand, T.vulcanus HspA chaperon protein was purified, which would be useful for removing aggregated proteins arising from the heat-treated thylakoid membrane to obtain the purified D1-D2 heterodimer proteins.