Purification of the infectious bovine rhinotracheitis virus alkaline deoxyribonuclease expressed in Escherichia coli.
Nucleotide sequence analysis within the unique long segment of the infectious bovine rhinotracheitis virus (IBRV) genome identified an open reading frame of 1461 base pairs whose deduced polypeptide of 487 amino acids exhibited homology to alkaline deoxyribonucleases of other herpesviruses. To determine whether this IBRV gene product has nuclease activity, the gene designated UL12 was inserted into the vector pET-28a(+) and expressed in Escherichia coli as an oligohistidine-tagged protein. Upon induction with isopropyl beta-D-thiogalactopyranoside E. coli BL21 (DE3)[pLysS] cells harboring this recombinant plasmid produced a 57-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using metal affinity chromatography resulted in a homogeneous preparation of this recombinant protein. The purified protein exhibited both exonuclease and endonuclease activities, each with an alkaline pH optimum.
Chung
Y T
YT
Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan.
Hsu
W
W
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003851:Deoxyribonucleases / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007242:Herpesvirus 1, Bovine / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D003851:Deoxyribonucleases