Cloning, expression, purification, and characterisation of the dUTPase encoded by the integrated Bacillus subtilis temperate bacteriophage SPbeta.
The cDNA encoding dUTPase, an enzyme catalysing the hydrolysis of dUTP to dUMP and pyrophosphate, from the integrated Bacillus subtilis temperate bacteriophage SPbeta has been cloned and over-expressed at high levels in Escherichia coli. The resulting recombinant dUTPase was purified to homogeneity in one step by phosphocellulose chromatography with a final yield of 700 mg pure crystallisable protein per litre of bacterial culture. The molecular mass of the 142 amino acid polypeptide was 16 kDa as judged by electrophoretic analysis and gel filtration chromatography revealed the enzyme to exist as a homotrimer in solution. Isoelectric focusing indicated the isoelectric point to be 7. Functionality of the purified recombinant dUTPase was proven by demonstrating catalytic activity towards the substrate dUTP. The optimal activity of SPbeta dUTPase proved to be dependent on the presence of divalent metal ions, with Mg(2+) conferring the highest activity.
Persson
Rebecca
R
York Structural Biology Laboratory, Department of Chemistry, University of York, Heslington, UK.
McGeehan
John
J
Wilson
Keith S
KS
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000821:virology*; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003460:Crystallization; D003856:Deoxyuracil Nucleotides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011755:Pyrophosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism
D002413:Cations, Divalent; D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase