dUTPase from herpes simplex virus type 1; purification from infected green monkey kidney (Vero) cells and from an overproducing Escherichia coli strain.
Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), widespread in nature with a crucial role in the nucleotide metabolism, catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. The enzyme from herpes simplex virus type 1 (HSV-1 dUTPase) was overproduced in Escherichia coli by using the T7 RNA polymerase expression system. The coding region of the HSV-1 dUTPase gene, UL 50, was positioned downstream of the promoter and the ribosome-binding site of the phage T7 gene 10 on the expression vector pET-3a. The resulting recombinant plasmid, pET-3a/UL50, was transformed into E. coli BL21(DE3)pLysS cells, conferring expression of HSV-1 dUTPase as 2-3% of the soluble protein inducible by isopropyl thiogalactoside. By chromatography on phosphocellulose and Mono S (Pharmacia LKB) columns a nearly homogeneous preparation of the enzyme with a high specific activity (49 mumol per minute per milligram) was obtained. The recombinant protein was compared with the native dUTPase similarly purified from HSV-1-infected Vero cells (African green monkey kidney fibroblasts). The two proteins showed the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amino-terminal sequences were found to be identical. The molecular mass (39 kDa) and the amino acid composition of the recombinant enzyme are also in accordance with predictions from the DNA sequence. Thus, the overproducing system described here appears suitable for providing HSV-1 dUTPase for detailed studies of molecular properties.
Björnberg
O
O
Department of Biochemistry, University of Lund, Sweden.
Bergman
A C
AC
Rosengren
A M
AM
Persson
R
R
Lehman
I R
IR
Nyman
P O
PO
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D017421:Sequence Analysis; D018139:Simplexvirus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D014709:Vero Cells; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000235:genetics
D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase