Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins

Ken Motohashi; Yuki Okegawa

doi:10.1016/j.pep.2014.07.001

Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins

Ken Motohashi, Yuki Okegawa

Research output: Contribution to journal › Article › peer-review

11 Citations (Scopus)

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.

Original language	English
Pages (from-to)	152-156
Number of pages	5
Journal	Protein Expression and Purification
Volume	101
DOIs	https://doi.org/10.1016/j.pep.2014.07.001
Publication status	Published - Sept 2014
Externally published	Yes

Keywords

Chloroplast
Disulfide bond
NADPH-dependent thioredoxin reductase
Redox regulation
Thioredoxin
Translation initiation codon

ASJC Scopus subject areas

Biotechnology

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10.1016/j.pep.2014.07.001

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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.",

keywords = "Chloroplast, Disulfide bond, NADPH-dependent thioredoxin reductase, Redox regulation, Thioredoxin, Translation initiation codon",

author = "Ken Motohashi and Yuki Okegawa",

note = "Funding Information: We thank Keisuke Yoshida and Toru Hisabori (Tokyo Institute of Technology) for peptide sequencing. This work was supported by JSPS KAKENHI Grant Numbers 22570037 (to K.M.) and 25650037 (to K.M.), and the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan (to K.M. and Y.O.).",

year = "2014",

month = sep,

doi = "10.1016/j.pep.2014.07.001",

language = "English",

volume = "101",

pages = "152--156",

journal = "Protein Expression and Purification",

issn = "1046-5928",

publisher = "Academic Press Inc.",

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T1 - Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins

AU - Motohashi, Ken

AU - Okegawa, Yuki

N1 - Funding Information: We thank Keisuke Yoshida and Toru Hisabori (Tokyo Institute of Technology) for peptide sequencing. This work was supported by JSPS KAKENHI Grant Numbers 22570037 (to K.M.) and 25650037 (to K.M.), and the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan (to K.M. and Y.O.).

PY - 2014/9

Y1 - 2014/9

N2 - 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.

AB - 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.

KW - Chloroplast

KW - Disulfide bond

KW - NADPH-dependent thioredoxin reductase

KW - Redox regulation

KW - Thioredoxin

KW - Translation initiation codon

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U2 - 10.1016/j.pep.2014.07.001

DO - 10.1016/j.pep.2014.07.001

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AN - SCOPUS:84905247175

SN - 1046-5928

VL - 101

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EP - 156

JO - Protein Expression and Purification

JF - Protein Expression and Purification

ER -

Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins

Abstract

Keywords

ASJC Scopus subject areas

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