Cloning, Expression, and Purification of Residues 495-624 of the Tanapox Viral Protein 84R (Isolate: TPV-Kenya)
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Smallpox was eradicated nearly thirty years ago, but growing concern over possible use of the variola virus in biological terrorism has reinvigorated the study of poxviruses, more specifically chordopoxviruses. Due to the high level of conservation in the genes encoding proteins required for essential viral functions in the Chordopoxvirinae, small poxviruses with mild clinical manifestation like tanapoxvirus are an attractive target for novel antiviral therapy against variola virus. DEAD-box helicases essential for gene expression were predicted to be present in the tanapoxvirus proteome and the C-terminal residues flanking the highly conserved helicase core were selected for further characterization. Following successful cloning and transformation, residues 495-624 of the tanapoxvirus 84R protein was expressed and the molecular weight of the protein was correctly identified by SDS-PAGE gel. A test of solubility on the pellet and supernatant helped indicate the protein as soluble. However, the results from protein purification, using a Ni-NTA Superflow resin column, were inconclusive, as the molecular weight of the protein could not be identified by SDS-PAGE gel. In the future, mass spectroscopy could be used in tandem with SDS-PAGE gel analysis to confirm the identity of the protein.