Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 797-800 |
| Number of pages | 4 |
| Journal | Biochemical Journal |
| Volume | 290 |
| Issue number | 3 |
| Publication status | Published - 1993 |
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ASJC Scopus subject areas
- Biochemistry
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Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli. / Polgár, L.; Erdélyi, F.; Hajnal, E.; Low, M.; Graf, L.; Korant, B. D.
In: Biochemical Journal, Vol. 290, No. 3, 1993, p. 797-800.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli
AU - Polgár, L.
AU - Erdélyi, F.
AU - Hajnal, E.
AU - Low, M.
AU - Graf, L.
AU - Korant, B. D.
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0027480987&partnerID=8YFLogxK
M3 - Article
C2 - 8457209
AN - SCOPUS:0027480987
VL - 290
SP - 797
EP - 800
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 3
ER -