Expression of mammalian DT-diaphorase in Escherichia coli: Purification and characterization of the expressed protein

Abstract

A full-length cDNA clone, pKK-DTD4, complementary to rat liver cytosolic DT-diaphorase [NAD(P)H:quinone oxidoreductase (EC 1.6.99.2)]mRNA was expressed in Escherichia coli. The pKK-DTD4 cDNA was obtained by extending the 5′-end sequence of a rat liver DT-diaphorase cDNA clone, pDTD55, to include an ATG initiation codon and the NH 2-terminal codons using polymerase chain reaction (PCR). Restriction sites for EcoRI and HindIII were incorporated at the 5′- and 3′-ends of the cDNA, respectively, by the PCR reaction. The resulting full-length cDNA was inserted into an expression vector, pKK2.7, at the EcoRI and HindIII restriction sites. E. coli strain AB1899 was transformed with the constructed expression plasmid, and DT-diaphorase was expressed under the control of the tac promoter. The expressed DT-diaphorase exhibited high activity of menadione reduction and was inhibited by dicumarol at a concentration of 10 −5 m. After purification by Cibacron Blue affinity chromatography, the expressed enzyme migrated as a single band on 12.5% sodium dodecyl sulfate-polyacrylamide gel with a molecular weight equivalent to that of the purified rat liver cytosolic DT-diaphorase. The purified expressed protein was recognized by polyclonal antibodies against rat liver DT-diaphorase on immunoblot analysis. It utilized either NADPH or NADH as electron donor at equal efficiency and displayed high activities in reduction of menadione, 1,4-benzoquinone, and 2,6-dichlorophenolindophenol which are typical substrates for DT-diaphorase. The expressed DT-diaphorase exhibited a typical flavoprotein spectrum with absorption peaks at 380 and 452 nm. Flavin content determination showed that it contained 2 mol of FAD per mole of the enzyme. Edman protein sequencing of the first 20 amino acid residues at the NH 2 terminus of the expressed protein indicated that the expressed DT-diaphorase is not blocked at the NH 2 terminus and has an alanine as the first amino acid. The remaining 19 amino acid residues at the NH 2 terminus were identical with those of the DT-diaphorase purified from rat liver cytosol.