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モトハシ タケシ
MOTOHASHI TAKESHI
本橋 健 所属 京都産業大学 生命科学部 先端生命科学科 職種 教授 |
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| 言語種別 | 英語 |
| 発行・発表の年月 | 2016/05 |
| 形態種別 | 研究論文 |
| 査読 | 査読あり |
| 標題 | Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system |
| 執筆形態 | その他 |
| 掲載誌名 | Protein Expression and Purification |
| 出版社・発行元 | Elsevier {BV} |
| 巻・号・頁 | 121,pp.46-51 |
| 著者・共著者 | Yuki Okegawa,Ken Motohashi |
| 概要 | Thioredoxins (Trxs) regulate the activity of target proteins in the chloroplast redox regulatory system. In vivo, a disulfide bond within Trxs is reduced by photochemically generated electrons via ferredoxin (Fd) and ferredoxin-thioredoxin reductase (FTR: EC 1.8.7.2). FTR is an alpha beta-heterodimer, and the beta-subunit has a 4Fe-4S cluster that is indispensable for the electron transfer from Fd to Trxs. Reconstitution of the light-dependent Fd/Trx system, including FTR, is required for the biochemical characterization of the Trx-dependent reduction pathway in the chloroplasts. In this study, we generated functional FTR by simultaneously expressing FTR-alpha and -beta subunits under the control of tandem T7 promoters in Escherichia coli, and purifying the resulting FTR complex protein. The purified FTR complex exhibited spectroscopic absorption at 410 nm, indicating that it contained the Fe-S cluster. Modification of the expression system and simplification of the purification steps resulted in improved FTR complex yields compared to those obtained in previous studies. Furthermore, the light-dependent Trx-reduction system was reconstituted by using Fd, the purified FTR, and intact thylakoids. (C) 2016 Elsevier Inc. All rights reserved. |
| DOI | 10.1016/j.pep.2016.01.005 |
| ISSN | 1046-5928/1096-0279 |
| PMID | 26773743 |
| Put Code(ORCID) | 21766029 |
| PermalinkURL | http://orcid.org/0000-0002-8414-2836 |