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Cell‐free production of active E. coli thioredoxin reductase and glutathione reductase
Author(s) -
Knapp Kurtis G,
Swartz James R
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(04)00025-0
Subject(s) - thioredoxin reductase , cofactor , cell free protein synthesis , flavin adenine dinucleotide , biochemistry , glutathione reductase , escherichia coli , reductase , chemistry , thioredoxin , enzyme , glutathione , flavin group , 7 dehydrocholesterol reductase , cell free system , protein biosynthesis , gene , glutathione peroxidase
Escherichia coli thioredoxin reductase (TR) and glutathione reductase (GR) are dimeric proteins that require a flavin adenine dinucleotide (FAD) cofactor for activity. A cell‐free protein synthesis (CFPS) reaction supplemented with FAD was used to produce TR at 760 μg/ml with 89% of the protein being soluble. GR accumulated to 521 μg/ml in a cell‐free reaction with 71% solubility. The TR produced was fully active with a specific activity of 1390 min −1 . The GR had a specific activity of 139 U/mg, which is significantly more active than reported for GR purified from cells. The specific activity for both TR and GR decreased without FAD supplementation. This research demonstrates that CFPS can be used to produce enzymes that are multimeric and require a cofactor.