PMID:1851868

Citation	Sankar, S and Porter, AG (1991) Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase. J. Virol. 65:2993-3000
Abstract	Encephalomyocarditis (EMC) virus RNA-dependent RNA polymerase was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), which allowed easy purification of the fusion protein by affinity chromatography on immobilized glutathione. Inclusion of a thrombin cleavage site between the GST carrier and the viral enzyme facilitated the release of purified mature EMC virus RNA polymerase from the GST carrier by proteolysis with thrombin. The purified recombinant enzyme has a molecular mass of about 52 kDa and is recognized by polyclonal immune serum raised against a peptide sequence corresponding to the C-terminal region of the protein. The recombinant enzyme comigrates with immunoprecipitated EMC virus RNA polymerase from infected mouse L929 cell extracts when run in parallel lanes on a sodium dodecyl sulfate-polyacrylamide gel. The enzyme exhibits rifampin-resistant, poly(A)-dependent poly(U) polymerase activity and RNA polymerase activity, which are both oligo(U) dependent. Template-size products are synthesized in in vitro reactions with EMC virus genomic RNA or globin mRNA. The availability of recombinant EMC virus RNA polymerase in a purified form will allow biochemical analysis of its role in the replication of the virus as well as structure-function studies of this unique class of enzyme.
Links	PubMed PMC240947
Keywords	Amino Acid Sequence; Antibodies, Viral/immunology; Base Sequence; Chromatography, Affinity; Drug Resistance, Microbial; Encephalomyocarditis virus/enzymology; Encephalomyocarditis virus/genetics; Encephalomyocarditis virus/growth & development; Escherichia coli/genetics; Gene Expression; Globins/genetics; Glutathione Transferase/genetics; Hydrolysis; Molecular Sequence Data; Plasmids; Precipitin Tests; RNA Replicase/genetics; RNA Replicase/immunology; RNA, Messenger/metabolism; Recombinant Fusion Proteins/biosynthesis; Recombinant Fusion Proteins/genetics; Rifamycins/pharmacology; Thrombin/metabolism; Virus Replication
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
EMCV:POLG		GO:0003968: RNA-directed RNA polymerase activity	ECO:0000314:		F		Figure 6.	complete CACAO 10476
EMCV:POLG	enables	GO:0003968: RNA-directed 5'-3' RNA polymerase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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