Expression, purification, and functional characterization of the DNA-binding domain of the herpes simplex virus type...

The origin-binding protein (UL9 protein) encoded by the herpes simplex virus type 1 (HSV-1) UL9 gene plays a critical role in the initiation of viral DNA replication. The 317-amino acid C-terminal DNA-binding domain (UL9-COOH) of the UL9 protein was expressed as a fusion protein to glutathione S-transferase in bacteria. Milligram quantities of the fusion protein were purified by differential ammonium sulfate precipitation and affinity chromatography over reduced glutathione agarose. The UL9-COOH moiety was cleaved from the fusion protein by proteolysis with thrombin. The purified UL9-COOH displayed specific and stable binding activity toward its DNA recognition sequences within the HSV-1 origin of DNA replication.