PMID:12071646

Citation	Persson, R, Nord, J, Roth, R and Nyman, PO (2002) dUTPase from Escherichia coli; high-level expression and one-step purification. Prep. Biochem. Biotechnol. 32:157-72
Abstract	The dut gene, which encodes Escherichia coli deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), has been recloned to increase overexpression of the enzyme and to enable simplification of the purification protocol into a one-step procedure. The gene was cloned into the vector pET-3a and expressed in E. coli BL21(DE3) pLysS under the control of a bacteriophage T7 promotor. Induction results in production of dUTPase corresponding to 60% of the extracted protein. Phosphocellulose chromatography at low pH was utilised for one-step purification, resulting in a homogenous preparation of the recombinant protein with a highly specific activity. The yield of purified enzyme is 500 mg per litre of bacterial culture, a significant increase compared to previously employed methods.
Links	PubMed Online version:10.1081/PB-120004128
Keywords	Base Sequence; Calibration; Cloning, Molecular; DNA Primers; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Escherichia coli/genetics; Genetic Vectors; Kinetics; Molecular Weight; Plasmids; Polymerase Chain Reaction; Pyrophosphatases/genetics; Pyrophosphatases/isolation & purification; Pyrophosphatases/metabolism; Recombinant Proteins/isolation & purification; Recombinant Proteins/metabolism
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