Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli

Yasumasa Komoda, Makoto Hijikata, Shinya Satoh, Shin Ichi Asabe, Koichi Kimura, Kunitada Shimotohno

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

Using as substrates a series of chimeric proteins containing various fragments of the hepatitis C virus precursor polyprotein between Escherichia coli maltose binding protein and dihydrofolate reductase, we analyzed the substrate requirements of hepatitis C viral serine proteinase (Cpro-2) for intermolecular polypeptide cleavage in E. coli. Cpro-2-dependent substrate cleavage was observed in E. coli cells simultaneously transformed with expression plasmids for the Cpro-2 molecule and substrate protein. The cleavage sites were estimated by determining the amino (N)-terminal amino acid sequences of dihydrofolate reductase-fused processed products purified partially by affinity chromatography from the lysates, indicating that cleavage occurred at sites identical to those observed in eukaryotic cells. Mutation analysis using the chimeric substrate indicated that the presence of cysteine and small uncharged residues at positions P1 and P1' respectively, of the putative cleavage site is necessary for cleavage and that acidic residues in the region upstream of the cleavage site are required for efficient cleavage.

Original languageEnglish
Pages (from-to)7351-7357
Number of pages7
JournalJournal of Virology
Volume68
Issue number11
Publication statusPublished - Nov 1994
Externally publishedYes

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Tetrahydrofolate Dehydrogenase
Hepatitis C virus
Serine Proteases
serine proteinases
Hepacivirus
dihydrofolate reductase
polypeptides
Maltose-Binding Proteins
Escherichia coli
Polyproteins
Peptides
Escherichia coli Proteins
Eukaryotic Cells
Hepatitis C
Affinity Chromatography
recombinant fusion proteins
hepatitis C
Cysteine
Amino Acid Sequence
Proteins

ASJC Scopus subject areas

  • Immunology

Cite this

Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli. / Komoda, Yasumasa; Hijikata, Makoto; Satoh, Shinya; Asabe, Shin Ichi; Kimura, Koichi; Shimotohno, Kunitada.

In: Journal of Virology, Vol. 68, No. 11, 11.1994, p. 7351-7357.

Research output: Contribution to journalArticle

Komoda, Y, Hijikata, M, Satoh, S, Asabe, SI, Kimura, K & Shimotohno, K 1994, 'Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli', Journal of Virology, vol. 68, no. 11, pp. 7351-7357.
Komoda, Yasumasa ; Hijikata, Makoto ; Satoh, Shinya ; Asabe, Shin Ichi ; Kimura, Koichi ; Shimotohno, Kunitada. / Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli. In: Journal of Virology. 1994 ; Vol. 68, No. 11. pp. 7351-7357.
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