Expression of trimeric human dUTP pyrophosphatase in Escherichia coli and purification of the enzyme.
In order to rapidly purify human dUTPase, a cDNA fragment that encodes the enzyme was subcloned and expressed using the Escherichia coli plasmid vector pGEX2T. The resulting plasmid expressed high levels of a glutathione S-transferase-dUTPase fusion protein following induction with IPTG. Affinity chromatography was used to purify the fusion protein, and dUTPase was then released from the fusion protein by thrombin treatment. The purified dUTPase has two additional vector-encoded residues at the amino terminus (gly-ser), but they have no apparent effect on the activity of the enzyme since the recombinant dUTPase has catalytic properties similar to those reported for dUTPase purified from human cells (32.3 U/mg, kcat = 25 s-1, Km = 2.6 microM). Enzyme activity was inhibited by 5-mercuri-dUTP and was shown to be sensitive to EDTA. Periodate-oxidized UTP had no effect on the activity of the enzyme, and dTTP caused only slight inhibition. The results of gel filtration experiments are consistent with a homotrimeric subunit composition for dUTPase. The ability to purify human dUTPase from E. coli should allow further characterization of the enzyme and provide material for the screening of potentially useful inhibitors.
Climie
S
S
Allelix Biopharmaceuticals Inc., Mississauga, Ontario, Canada.
Lutz
T
T
Radul
J
J
Sumner-Smith
M
M
Vandenberg
E
E
McIntosh
E
E
D001483:Base Sequence; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase