RELEASE OF A 22-KDA PROTEIN DERIVED FROM THE AMINO-TERMINAL DOMAIN OF THE 49-KDA NLA OF TURNIP MOSAIC POTYVIRUS IN ESCHERICHIA-COLI

The coding region for the precursor 6K-small nuclear inclusion a (NIa) protein and for the NIa protein of turnip mosaic potyvirus (TuMV) were introduced into the plasmid pET-11d for high-level expression in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analyses of E. coli proteins showed that the Me protein underwent endoproteolysis and released a 22-kDa polypeptide. NH2-terminal amino acid sequencing of the recombinant 22-kDa protein was performed and was identical to the predicted amino end of the Nis protein. Site-directed mutagenesis confirmed that the hydrolysis was associated with the Nla proteolytic activity and that the proteinase recognized a Glu residue within en amino acid sequence found in the Nla protein which fitted the TuMV consensus cleavage site sequence. Fusion of the 6K protein with the Nla protein partially inhibited the hydrolytic reaction. The recombinant 22-kDa protein is likely the VPg of TuMV. © 1992.