Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli.
Poliovirus protease 3C is a cysteine enzyme that is essential for the processing of the viral precursor polyprotein containing structural proteins and enzymes, including the protease itself. We have constructed the plasmid pSD/PV3C which produced protease 3C as inclusion bodies when expressed in Escherichia coli. In addition to the full-length protease, a truncated form was also generated, starting from an internal initiation site (Met-27). The enzyme was renatured by dilution of a 6 M guanidinium chloride solution of the inclusion bodies, and the proteins were precipitated from the diluted solution with ammonium sulphate. By extracting the precipitate with a buffer solution, the full-length enzyme could be completely separated from its N-terminally truncated form. Size-exclusion chromatography of the extracted protease 3C resulted in an active enzyme which appeared homogeneous by SDS/PAGE. For measuring the activity of the protease, a spectrofluorimetric method was devised to monitor the hydrolysis continuously, which is simpler and more precise than the h.p.l.c. technique used previously.
Polgár
L
L
Institute of Enzymology, Hungarian Academy of Sciences, Budapest.
Erdélyi
F
F
Hajnal
E
E
Löw
M
M
Gráf
L
L
Korant
B D
BD
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins
D009842:Oligopeptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases