Processing of hepatitis C viral polyprotein in Escherichia coli.
Two proteinase activities, encoded by hepatitis C virus (HCV), Cpro-1 and Cpro-2. Cpro-1 and Cpro-2 appear to process the precursor polyprotein from which they originate. Mutant HCV polypeptides containing the region for these proteinases were produced in Escherichia coli as fusion proteins. The N- and C-terminal ends of the HCV polypeptides were fused with the E. coli maltose-binding protein (MBP) and E. coli dihydrofolate reductase (DHFR), respectively. The proteinase activities cleaved the fusion polypeptides by the same processing pathway used in eukaryotic protein production systems. The N-terminal amino acid (aa) sequences of the processed fusion proteins were determined. A comparison of those N-terminal sequences with the aa sequence of the HCV precursor polyprotein showed that the N-terminal and C-terminal cleavage sites of p70(NS3), one of the HCV nonstructural (NS) proteins, were the same as those identified in other processing studies: cleavages were estimated to be between aa 1026 and 1027 and between aa 1657 and 1658 of the HCV precursor protein, which are known to be cleaved by Cpro-1 and Cpro-2, respectively. Cpro-1 and Cpro-2 both functioned in E. coli and possessed authentic characteristic features.
Komoda
Y
Y
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Hijikata
M
M
Tanji
Y
Y
Hirowatari
Y
Y
Mizushima
H
H
Kimura
K
K
Shimotohno
K
K
D018528:ATP-Binding Cassette Transporters; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D016174:Hepacivirus / Q000201:enzymology*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C461599:maltose transport system, E coli; D013762:Tetrahydrofolate Dehydrogenase; D010450:Endopeptidases; C084815:proteinase Cpro-1, hepatitis C virus; C084816:proteinase Cpro-2, hepatitis C virus