Purification, properties, and mutagenesis of poliovirus 3C protease》.pdf

VIROLOGY 185,140-150(1991) Purification, Properties, and Mutagenesis of Poliovirus 3C Protease ELLEN Z. BAUM, GERALDINE A. BEBERNITZ, OLGA PALANT, THOMAS MUELLER, AND STEPHEN .I. PLOTCH Molecular Biology Section, Lederle Laboratories, Medical Research Division, American Cyanamid Company, Pearl River, New York 10965 Received February 12, 199 1;accepted July 22, 199 1 Poliovirus protease 3C, type 1 Mahoney strain, was expressed in Escherichia co/i under phage T7 promoter control and purified to homogeneity from resolubilized inclusion bodies. The renatured protein was as enzymatically active as the protease found in the soluble portion of the bacterial lysate. Proteolytic activity was assayed using as substrate either [%]methionine-labeled recombinant poliovirus proteins 2C3AB or a truncated version of 3ABC, or synthetic peptide 16-mers corresponding to the cleavage sites at 2C/3A and 3A/3B. Poliovirus protein 3CD (protease-polymer- ase) was also expressed in bacteria. About 25% of this protein apparently autodigested in viva, releasing immunopreci- pitable protein 3D (polymerase). No further autodigestion of 3CD could be detected in vitro, nor could addition of purified protein 3C effect digestion in trans. Both the serine protease inhibitors PMSF, TPCK, and 3,4-dichloroisocou- marin, and the cysteine protease inhibitors cystatin and zinc, were effective inhibitors of the 3C protease. Six new