Functional expression of the recombinant ATPase of orf virus.
Nucleotide sequence analysis has indicated that the A32L gene of orf virus can encode an ATPase (Chan et al. in Gene 432:44-53, 2009). In this work, we cloned the A32L gene into a prokaryotic expression vector, and the recombinant protein was expressed and purified. The antigenicity of recombinant ATPase was examined by immunoblotting, and its identity was confirmed by mass spectrometry. The ATP hydrolysis function of the purified recombinant protein was examined, and our results showed that it exhibited the ATPase activity. Similar to other viral ATPases, the ATPase of orf virus remained active in the presence of different divalent ions; nevertheless, unlike other viral ATPases, our recombinant ATPase exhibited similar enzymatic activity in reaction buffers of different pH.
Lin
Fong-Yuan
FY
Department of Veterinary Medicine, College of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan.
Chan
Kun-Wei
KW
Wang
Hsien-Chi
HC
Hsu
Wei-Li
WL
Wong
Min-Liang
ML
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009923:Orf virus / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D002413:Cations, Divalent; D003067:Coenzymes; D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases