title: Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins.
abstract: Plant redox-related proteins were overexpressed using a genetic codon substitution downstream of the translation initiation codon. This method significantly improved recombinant protein expression levels of Arabidopsis chloroplastic thioredoxins and cytosolic nicotinamide adenine dinucleotide phosphate (NADPH)-dependent thioredoxin reductase (E.C. 1.8.1.9) in Escherichia coli. Using these proteins, the in vitro chloroplastic thioredoxins-reduction system was reconstituted in an NADPH-dependent manner. This system could convert the five classes of chloroplastic Arabidopsis thioredoxins and two chloroplastic Spinach thioredoxins to their reduced forms, independent of dithiothreitol and the photosynthetic electron transport system.
authors.affiliation: Department of Bioresources 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
authors.affiliation: Department of Bioresources 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
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology*; D001483:Base Sequence; D054479:Chloroplast Thioredoxins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004579:Electron Transport / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010084:Oxidation-Reduction; D010788:Photosynthesis / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics; D018724:Spinacia oleracea / Q000201:enzymology*; D013880:Thioredoxin-Disulfide Reductase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
chemical_list: D054479:Chloroplast Thioredoxins; D010940:Plant Proteins; D011994:Recombinant Proteins; D013880:Thioredoxin-Disulfide Reductase
keywords: Chloroplast; Disulfide bond; NADPH-dependent thioredoxin reductase; Redox regulation; Thioredoxin; Translation initiation codon