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

光系统II反应中心亚基D1-D2异源二聚体催化水裂解和出氧反应。为了建立一种分离异二聚体的方法，通过rps12介导的基因替代构建重组长聚球菌，将嗜热蓝藻vulcanus Thermasynechococcus vulcanus D1和D2蛋白在嗜热长聚球菌中表达。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，从这些蓝藻中制备了类囊体膜蛋白，并使用T.vulcanusD1-1蛋白特异性抗体进行了Western blotting分析。虽然在GRPS201和GRPS220菌株中均检测到D1蛋白，但D1-1的大小比原产T.vulcanus的D1-1大2kDa，说明c端16个氨基酸未加工。另一方面，我们纯化了T.vulcanus HspA伴侣蛋白，这将有助于去除热处理的类囊体膜上产生的聚集蛋白，得到纯化的D1-D2异二聚体蛋白。

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.