2011年7月
A heterogeneous tag-attachment to the homodimeric type 1 photosynthetic reaction center core protein in the green sulfur bacterium Chlorobaculum tepidum
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
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- 巻
- 1807
- 号
- 7
- 開始ページ
- 803
- 終了ページ
- 812
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.bbabio.2011.03.007
- 出版者・発行元
- ELSEVIER SCIENCE BV
The 6xHis-tag-pscA gene, which was genetically engineered to express N-terminally histidine (His)-tagged PscA, was inserted into a coding region of the recA gene in the green sulfur bacterium Chlorobaculum tepidum (C. tepidum). Although the inactivation of the recA gene strongly suppressed a homologous recombination in C. tepidum genomic DNA, the mutant grew well under normal photosynthetic conditions. The His-tagged reaction center (RC) complex could be obtained simply by Ni(2+)-affinity chromatography after detergent solubilization of chlorosome-containing membranes. The complex consisted of three subunits, PscA, PscB, and PscC, in addition to the Fenna-Matthews-Olson protein, but there was no PscD. Low-temperature EPR spectroscopic studies in combination with transient absorption measurements indicated that the complex contained all intrinsic electron transfer cofactors as detected in the wild-type strain. Furthermore, the LC/MS/MS analysis revealed that the core protein consisted of a mixture of a His-/His-tagged PscA homodimer and a non-/His-tagged PscA heterodimer. The development of the pscA gene duplication method presented here, thus, enables not only a quick and large-scale preparation of the RC complex from C. tepidum but also site-directed mutagenesis experiments on the artificially incorporated 6xHis-tag-pscA gene itself, since the expression of the authentic PscA/PscA homodimeric RC complex could complement any defect in mutated His-tagged PscA. This method would provide an invaluable tool for structural and functional analyses of the homodimeric type 1 RC complex. (C) 2011 Elsevier B.V. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.bbabio.2011.03.007
- ISSN : 0005-2728
- ORCIDのPut Code : 49957806
- Web of Science ID : WOS:000291518400004