A simple purification and fluorescent assay method of the poliovirus 3C protease searching for specific inhibitors.
Picornaviruses such as poliovirus, foot-and-mouth disease virus, and encephalomyocarditis virus produce their proteins by translating their genomic RNA, injected within the host cell, into a precursor polyprotein, which is then subjected to precise processing. The polyprotein is cleaved into mature proteins predominantly by the viral 3C protease. A simple purification and assay method for poliovirus 3C protease for use for screening for inhibitors of the 3C protease is described. A poliovirus cDNA fragment containing the 3C protease coding region was inserted into pET22b vector and expressed in Escherichia coli. The His-tagged protein (3CD'-His) was purified by a Ni-affinity column and the activity of the purified enzyme was measured by a fluorescent assay with a fluorogenic substrate containing the 3C-specific cleavage site, MocAc-MEALFQGPLQY-Dnp. The kinetic parameters calculated from the Lineweaver-Burk plot and the effects of inhibitors showed that E. coli expression with His tag and the assay using the fluorogenic substrate are efficient, simple and sensitive methods for purifying the 3C protease, and measuring its activity.
Hata
S
S
Laboratory of Molecular Structure and Function, Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Sato
T
T
Sorimachi
H
H
Ishiura
S
S
Suzuki
K
K
D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D005966:Glucuronidase / Q000378:metabolism; D006367:HeLa Cells; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017955:Poliovirus / Q000201:enzymology* / Q000235:genetics; D020815:Polyproteins / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins
D005456:Fluorescent Dyes; D020815:Polyproteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D014764:Viral Proteins; D006639:Histidine; D005966:Glucuronidase; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases