dUTPase from the retrovirus equine infectious anemia virus: high-level expression in Escherichia coli and purification.
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, and plays important roles in nucleotide metabolism and DNA replication. The dUTPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymerase expression system. The recombinant vector (pET-3a/EDU), constructed by mutagenic PCR, was transformed into E. coli BL21 (DE3) pLysS cells, resulting in expression of EIAV dUTPase at about 40% of the extracted protein. This level of overproduction is very high compared to previous reports on heterologous expression of dUTPases in E. coli. A one-step purification procedure using phosphocellulose chromatography results in a homogeneous preparation of the enzyme in a yield of 45 mg liter-1 of bacterial culture. The purified EIAV dUTPase, run on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows an apparent molecular mass of 15.1 kDa in accordance with the gene structure. The isoelectric point (pI) was determined to 5.6. Gel filtration under nondenaturating conditions gives a retention volume corresponding to a molecular mass of 40.6 kDa, suggesting a trimeric organization of the enzyme. The amino acid composition and amino-terminal sequence of the recombinant dUTPase are in agreement with predictions from the DNA sequence.
Bergman
A C
AC
Department of Biochemistry, Lund University, Sweden.
Björnberg
O
O
Nord
J
J
Rosengren
A M
AM
Nyman
P O
PO
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D003001:Cloning, Molecular; D003856:Deoxyuracil Nucleotides / Q000378:metabolism*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004860:Infectious Anemia Virus, Equine / Q000201:enzymology*; D007525:Isoelectric Focusing; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D013053:Spectrophotometry
D000596:Amino Acids; D003856:Deoxyuracil Nucleotides; D011994:Recombinant Proteins; C027078:deoxyuridine triphosphate; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase