Bacterial expression and analysis of cleavage activity of HCV serine proteinase using recombinant and synthetic substrate.
HCV encoding serine proteinase was expressed in E. coli as a fused form with maltose binding protein (MBP) and a six histidine tag. The enzyme was partially purified by using affinity chromatography for these fused peptides. Proteolytic cleavage activity of the partially purified enzyme was detected by means of an assay using both a recombinant protein and a synthetic peptide substrate which had an amino acid sequence corresponding to the most efficient cleavage site in vivo, the NS5A-NS5B junction. The cleavage occurred at the same site that was reported before.
Kakiuchi
N
N
HQL Research Labs. Sumitomo Metal Industries Ltd., Kyoto, Japan.
Hijikata
M
M
Komoda
Y
Y
Tanji
Y
Y
Hirowatari
Y
Y
Shimotohno
K
K
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000096:biosynthesis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D016174:Hepacivirus / Q000378:metabolism*; D006639:Histidine; D008320:Maltose / Q000378:metabolism; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D016324:Sequence Tagged Sites; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C461599:maltose transport system, E coli; D006639:Histidine; D008320:Maltose; D012697:Serine Endopeptidases