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异二聚体催化水分解和放氧反应。为了建立分离异源二聚体的方法，利用rps 12基因置换法构建了重组细长聚球藻，在中温聚球藻中表达了高温蓝细菌Thermasynechococcus vulcanus的D1和D2蛋白。转化携带rps 12 R-43突变的GRPS 1以获得GRPS 201和GRPS 220菌株。GRPS 201携带来自T. vulcanus的替换的psbA 1基因，而GRPS 220携带来自T.vulcanus的替换的psbA 1和psbD 1基因。从这些蓝细菌制备类囊体膜蛋白，并使用T.vulcanusD1-1蛋白特异性抗体通过Western印迹分析。虽然在GRPS 201和GRPS 220菌株中检测到D1蛋白，但D1-1的大小比来自T.vulcanus的天然D1-1大2kDa，表明C端16个氨基酸未被加工。另一方面，纯化了T.vulcanus HspA伴侣蛋白，这将有助于去除从热处理的类囊体膜产生的聚集蛋白，以获得纯化的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.