dUTPase from Escherichia coli; high-level expression and one-step purification.
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.
Persson
Rebecca
R
Department of Chemistry, University of York, Heslington, UK. persson@ysbl.york.ac.uk
Nord
Johan
J
Roth
Robert
R
Nyman
Per Olof
PO
D001483:Base Sequence; D002138:Calibration; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase