PMID:14960309

Citation	Knapp, KG and Swartz, JR (2004) Cell-free production of active E. coli thioredoxin reductase and glutathione reductase. FEBS Lett. 559:66-70
Abstract	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 microg/ml with 89% of the protein being soluble. GR accumulated to 521 microg/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.
Links	PubMed Online version:10.1016/S0014-5793(04)00025-0
Keywords	Cell-Free System; Cloning, Molecular/methods; Escherichia coli Proteins/biosynthesis; Flavin-Adenine Dinucleotide; Glutathione Reductase/biosynthesis; Glutathione Reductase/isolation & purification; Glutathione Reductase/metabolism; Kinetics; Recombinant Fusion Proteins; Solubility; Thioredoxin-Disulfide Reductase/biosynthesis; Thioredoxin-Disulfide Reductase/isolation & purification; Thioredoxin-Disulfide Reductase/metabolism
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