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

携带耐热光系统 II 的重组蓝藻的析氧最小复合物的纯化

• 批准号: 15560682
• 负责人: MATSUOKA Masayoshi
• 金额: $ 1.98万
• 依托单位: Sojo University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15560682/
• 关键词: photosystem II; cyanobacteria; gene replacement; D1-D2 heterodimer; thermostability; Synechococcus elongatus; Thermosynechococcus vulcanus; oxygen evolution; 熱安定性; 熱凝集; シャペロン; 熱安定化

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.

光系统II反应中心亚基，D1-D2异二聚体催化水分解和氧气放出反应。为了建立分离异二聚体的方法，通过 rps12 介导的基因替换构建重组 S.elongatus，在嗜温蓝细菌 Thermasynechocococcus vulcanus 中表达 D1 和 D2 蛋白，在嗜温的 S.elongatus 中表达。将携带rps12R-43突变的GRPS1转化以获得GRPS201和GRPS220菌株。 GRPS201 携带替换的 psbA1 基因，而 GRPS220 携带来自 T.vulcanus 的替换 psbA1 和 psbD1 基因。从这些蓝藻中制备类囊体膜蛋白，并使用 T.vulcanusD1-1 蛋白特异性抗体通过蛋白质印迹进行分析。尽管在 GRPS201 和 GRPS220 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 个氨基酸没有被加工。另一方面，纯化了T.vulcanus HspA伴侣蛋白，这可用于去除热处理类囊体膜上产生的聚集蛋白，以获得纯化的D1-D2异二聚体蛋白。

项目成果

Takahama K., Matsuoka, M., Nagahama, K., Ogawa, T.: "High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene"Plant Cell Physiol.. Vol.45,No.3(in press). (2004)

Takahama K.、Matsuoka, M.、Nagahama, K.、Okawa, T.：“使用异源 rps12 基因在蓝细菌中进行高频基因替换”Plant Cell Physiol.. Vol.45，No.3（出版中）。

「研究成果報告書概要(欧文)」より

摘自《研究结果报告摘要（欧洲）》

• 发表时间: 2006
• 期刊: Seibutsu Butsuri 46(1)
• 作者: Yasushi Shigeri;Keiko Shimamoto
• 通讯作者: Keiko Shimamoto

High-frequency gene replacement in cyanobacteria using a heterologous rpsl2 gene

使用异源 rpsl2 基因在蓝藻中进行高频基因替换

• 发表时间: 2004
• 期刊: Plant Cell Physiol. Vol.45, No.3
• 作者: Takahama;K.;Matsuoka;M.;Nagahama;K.;Ogawa;T.
• 通讯作者: T.

High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene

使用异源 rps12 基因在蓝藻中进行高频基因替换

• 发表时间: 2004
• 期刊: Plant Cell Physiol. Vol.45,No.3
• 作者: Takahama;K.;Matsuoka;M.;Nagahama;K.;Ogawa;T.
• 通讯作者: T.