Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli. 793 18

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.
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PMID:Processing of hepatitis C viral polyprotein in Escherichia coli. 805 35