Histidine codons appended to the gene encoding the RPO22 subunit of vaccinia virus RNA polymerase facilitate the isolation and purification of functional enzyme and associated proteins from virus-infected cells.
Vaccinia virus encodes a eukaryotic-like RNA polymerase composed of two large and six small subunit protein species. A replication-competent virus with 10 histidine codons added to the single endogenous J4R open reading frame was constructed. The altered migration of the 22-kDa subunit of RNA polymerase on SDS-polyacrylamide gel electrophoresis confirmed that J4R encoded the RPO22 subunit and that the mutant virus was genetically stable. The histidine-tagged RNA polymerase bound quantitatively to metal-affinity resins and was eluted in an active form upon addition of imidazole. Glycerol gradient sedimentation of the eluted fraction indicated that most of the RPO22 in infected cells is associated with RNA polymerase. Using stringent washing conditions, metal-affinity chromatography resulted in a several hundred-fold increase in RNA-polymerase-specific activity, and substantially pure enzyme was obtained with an additional conventional chromatography step. When mild conditions were used for washing the metal-affinity resin, the vaccinia virus-encoded capping enzyme, early transcription factor, and nucleoside triphosphate phosphohydrolase I specifically co-eluted with the tagged RNA polymerase, consistent with their physical association. The ability to selectively bind RNA polymerase to an affinity column provided a simple and rapid method of concentrating and purifying active enzyme and protein complexes.
Katsafanas
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National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20892-0445, USA.
Moss
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D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity; D003062:Codon; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005814:Genes, Viral; D006367:HeLa Cells; D006639:Histidine; D006801:Humans; D008670:Metals; D011995:Recombination, Genetic; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D003062:Codon; D008670:Metals; D014764:Viral Proteins; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; C119739:RPO22 protein, Vaccinia virus