PURIFICATION OF THE INFECTIOUS BOVINE-RHINOTRACHEITIS VIRUS ALKALINE DEOXYRIBONUCLEASE EXPRESSED IN ESCHERICHIA-COLI

Nucleotide sequence analysis within the unique long segment of the inf ectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amin o acids exhibited homology to alkaline deoxyribonucleases of other her pesviruses. To determine whether this IBRV gene product has nuclease a ctivity, the gene designated UL12 was inserted into the vector pET-28a (+) and expressed in Escherichia coli as an oligohistidine-tagged prot ein. Upon induction with isopropyl beta-D-thiogalactopyranoside E. col i BL21 (DE3)[pLysS] cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with t he prediction from the nucleotide sequence. A one-step purification pr ocedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein. The purified protein exhibite d both exonuclease and endonuclease activities, each with an alkaline pH optimum.