Minimization of thermostable water-splitting machinery of photosystem II by subunit gene replacement

光化学裂水反应所需的最小单元是光系统II(PSII)反应中心的异源二聚体，由D1和D2蛋白组成。我们研究了从嗜热蓝细菌D1/D2异源二聚体中分离表达耐高温DL/D2蛋白的细长聚球藻PCC7942的可能性。为此，我们利用rps12介导的基因置换技术，将S细长线虫的d1和d2基因的ORF替换为T硫杆菌的ORF，这是一种不受耐药性标记可获得性限制的新技术。在PSBB基因编码的CP47蛋白中引入C-末端组氨酸标签，以促进PSII复合体的纯化。由此产生的psbB突变株对psbB基因是异质性的，因此通过在psbT基因上游插入额外的启动子拷贝来激活psbB基因下游的psbT基因。此外，还克隆了一段包含psbB-psbT区的DNA片段，以提高重组频率。利用所构建的psbB突变株，通过镍亲和层析，有效地从PSII复合体中分离纯化了d1/d2杂二聚体。

A minimum unit required for photochemical water-splitting reaction is a heterodimer consisting of D1 and D2 proteins in reaction center of photosystem II (PSII). We investigated possible isolation of D 1/D2 heterodimer from mesophilic cyanobacterium, Synechococcus elongatus PCC 7942 expressing thermostable Dl/D2 proteins from a thermophile Thermosynechococcus vulcanus. To this end, we replaced ORFs of D1 and D2 genes in S elongatus with those from T vulcanus via rps12-mediated gene replacement method, a newly developed technology without limitation of the availability of drug-resistance markers. A C-terminal histidine tag was introduced into CP47 protein encoded by psbB gene to facilitate the purification of PSII complex. The resultant psbB mutant strain was heterogeneous for psbB gene, so that a psbT gene downstream the psbB gene was activated by inserting an extra copy of promoter upstream the psbT gene. In addition, a long stretch of DNA encompassing the psbB psbTregion was cloned to enhance the recombination frequency between plasmid and chromosome. Using psbB mutant strains thus created, Dl/D2 heterodimer was efficiently purified from PSII complex via Ni-affinity chromatography.