Application of a single-plasmid vector for mutagenesis and high-level expression of thioredoxin reductase and its use to examine flavin cofactor incorporation.
Thioredoxin reductase from Escherichia coli is a dimeric enzyme containing one FAD and one redox-active disulfide per monomer and catalyzes the transfer of electrons from NADPH to thioredoxin, which subsequently performs several important cellular functions. To overcome problems with site-directed mutagenesis and low expression, the thioredoxin reductase gene was adapted for use in the plasmid vector pSL350 (Brosius, J., Methods Enzymol. 216, 469-483, 1992), which is designed both for protein expression and for production of single-stranded template DNA for mutagenesis, and examined expression of wild-type thioredoxin reductase under different growth conditions. In the absence of IPTG inducer, expression of thioredoxin reductase in saturated cultures accounts for 19% of the soluble protein, and with 1 mM IPTG expression increases to 61%. Some of the thioredoxin reductase is expressed as apoenzyme with the amount of apoenzyme increasing at higher IPTG concentrations, accounting for as high as 68% of the total thioredoxin reductase expressed. The apoenzyme in cell extracts is activated rapidly by addition of FAD, indicating correct folding of the enzyme in the absence of cofactor. Purification of wild-type thioredoxin reductase from the new system yielded 189 mg of enzyme from a 300-ml uninduced culture. The new plasmid was also used to generate an N155Y mutant which is purified and partially characterized.
Mulrooney
S B
SB
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606, USA.
D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007544:Isopropyl Thiogalactoside; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D007544:Isopropyl Thiogalactoside; D013880:Thioredoxin-Disulfide Reductase