Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli

L. Polgár, F. Erdélyi, E. Hajnal, M. Low, L. Graf, B. D. Korant

Research output: Contribution to journalArticle

4 Citations (Scopus)

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 languageEnglish
Pages (from-to)797-800
Number of pages4
JournalBiochemical Journal
Volume290
Issue number3
Publication statusPublished - 1993

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Escherichia coli
Enzymes
Peptide Hydrolases
Inclusion Bodies
Polyproteins
Size exclusion chromatography
Guanidine
Ammonium Sulfate
Dilution
Gel Chromatography
Cysteine
Precipitates
Polyacrylamide Gel Electrophoresis
Hydrolysis
Buffers
Proteins
Plasmids
3C proteases
Processing

ASJC Scopus subject areas

  • Biochemistry

Cite this

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 journalArticle

Polgár, L. ; Erdélyi, F. ; Hajnal, E. ; Low, M. ; Graf, L. ; Korant, B. D. / Separation of native and truncated forms of poliovirus protease 3C produced in Escherichia coli. In: Biochemical Journal. 1993 ; Vol. 290, No. 3. pp. 797-800.
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