title: Expression of spinach ferredoxin-thioredoxin reductase using tandem T7 promoters and application of the purified protein for in vitro light-dependent thioredoxin-reduction system.
abstract: 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 αβ-heterodimer, and the β-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-α and -β 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.
authors.affiliation: Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Okegawa
authors.affiliation: Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan. Electronic address: motohas@cc.kyoto-su.ac.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Motohashi
mesh_terms: D054479:Chloroplast Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002736:Chloroplasts / Q000737:chemistry / Q000378:metabolism; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000096:biosynthesis* / Q000235:genetics; D008027:Light; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics; D010788:Photosynthesis; D018724:Spinacia oleracea / Q000201:enzymology
chemical_list: D054479:Chloroplast Thioredoxins; D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D010088:Oxidoreductases; C021588:ferredoxin-thioredoxin reductase
keywords: Co-expression; Ferredoxin-thioredoxin reductase; Photosynthetic electron transport; Reconstitution; Redox-regulation; Thioredoxin