cis- and trans-cleavage activities of poliovirus 2A protease expressed in Escherichia coli.
The poliovirus protease, 2Apro, was produced in Escherichia coli from plasmids that encode a fusion protein consisting of the N-terminal portion of the bacterial TrpE protein linked to poliovirus 2Apro. This fusion protein underwent efficient autocatalytic cleavage at the N terminus of 2Apro, generating the mature protease. Extracts of bacteria expressing 2Apro induced the specific cleavage of the p220 subunit of the eukaryotic translation initiation factor 4F, similar to the 2Apro-mediated reaction that occurs in poliovirus-infected HeLa cells. A portion of the poliovirus polyprotein containing the 2Apro cleavage site at the P1/P2 junction was produced by translation of cDNA transcripts in rabbit reticulocyte lysates and then tested as a substrate for 2Apro-mediated cleavage. The protein was partially cleaved by 2Apro in trans. Finally, a 16-amino-acid synthetic peptide, representing the P1/P2 junction sequence, was analyzed as a substrate for 2Apro. The peptide was labeled with fluorescein at a lysine residue to facilitate its detection. Recombinant 2Apro cleaved the synthetic peptide into two half-peptide molecules which were resolved by high-pressure liquid chromatography. Direct sequence analysis of the isolated peptide products demonstrated that cleavage occurred at the expected tyrosine-glycine pair. A rapid cleavage assay for 2Apro activity on the synthetic peptide was developed, using separation of the fluorescein-labeled 8-amino-acid product from the 16-residue substrate by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels.
Alvey
J C
JC
Department of Cellular Biology, University of Utah, School of Medicine, Salt Lake City 84132.
Wyckoff
E E
EE
Yu
S F
SF
Lloyd
R
R
Ehrenfeld
E
E
D001483:Base Sequence; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017955:Poliovirus / Q000201:enzymology / Q000235:genetics*; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus