Alvey, J.C., Wyckoff, E.E., Yu, S.F., Lloyd, R., Ehrenfeld, E. 1991. cis- and trans-cleavage activities of poliovirus 2A protease expressed in Escherichia coli. Journal of Virology. 65(11):6077-6083.
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.
