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 play s important roles in nucleotide metabolism and DNA replication. The dU TPase gene of the retrovirus equine infectious anemia virus (EIAV) was cloned and overexpressed in Escherichia coli using the T7 RNA polymer ase expression system. The recombinant vector (pET-3a/EDU), constructe d by mutagenic PCR, was transformed into E. coli BL21(DE3) pLysS cells , resulting in expression of EIAV dUTPase at about 40% of the extracte d protein, This level of overproduction is very high compared to previ ous reports on heterologous expression of dUTPases in E. coli. A one-s tep purification procedure using phosphocellulose chromatography resul ts in a homogeneous preparation of the enzyme in a yield of 45 mg lite r(-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 (pr) was determined to 5.6. Gel filtration under no ndenaturating conditions gives a retention volume corresponding to a m olecular mass of 40.8 kDa, suggesting a trimeric organization of the e nzyme. The amino acid composition and amino-terminal sequence of the r ecombinant dUTPase are in agreement with predictions from the DNA sequ ence. (C) 1995 Academic Press, Inc.