Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity.
The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domain derived from the 3' end of gag, fused to 154 residues encoded by the 5' region of the pro open reading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted by site-directed mutagenesis to allow the read-through from the gag into the pro open reading frame. The obtained insert was then cloned into pGEX-2T, a plasmid containing the glutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30) was isolated by affinity chromatography on a glutathione-Sepharose 4B column, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparent Km for dUTP was 28 microM. The enzyme was inhibited by EDTA, but its effect could be reversed by Mg2+ and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPase coding region.
Köppe
B
B
Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research and Development Center, Maryland 21702-1201.
Menéndez-Arias
L
L
Oroszlan
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002614:Chelating Agents / Q000494:pharmacology; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015684:Gene Products, pol / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000201:enzymology*; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016364:Reading Frames; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013379:Substrate Specificity; D014758:Viral Core Proteins / Q000187:drug effects / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002413:Cations, Divalent; D002614:Chelating Agents; D015683:Gene Products, gag; D015684:Gene Products, pol; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D011755:Pyrophosphatases; C020020:dUTP pyrophosphatase