Expression, purification, and properties of recombinant encephalomyocarditis virus RNA-dependent RNA polymerase.
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.
Sankar
S
S
Institute of Molecular and Cell Biology, National University of Singapore.
Porter
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AG
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000276:immunology; D001483:Base Sequence; D002846:Chromatography, Affinity; D004352:Drug Resistance, Microbial; D004686:Encephalomyocarditis virus / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005914:Globins / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010957:Plasmids; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012294:Rifamycins / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism; D014779:Virus Replication
D000914:Antibodies, Viral; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D012294:Rifamycins; D005914:Globins; D005982:Glutathione Transferase; D012324:RNA-Dependent RNA Polymerase; D013917:Thrombin