SUBSTRATE REQUIREMENTS OF HEPATITIS-C VIRUS SERINE PROTEINASE FOR INTERMOLECULAR POLYPEPTIDE CLEAVAGE IN ESCHERICHIA-COLI

被引:40
作者
KOMODA, Y
HIJIKATA, M
SATO, S
ASABE, SI
KIMURA, K
SHIMOTOHNO, K
机构
[1] NATL CANC CTR,RES INST,DIV VIROL,CHUO KU,TOKYO 104,JAPAN
[2] SCI UNIV TOKYO,FAC SCI & TECHNOL,DEPT APPL BIOL SCI,NODA,CHIBA 278,JAPAN
关键词
D O I
10.1128/JVI.68.11.7351-7357.1994
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 aminoacid 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.
引用
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页码:7351 / 7357
页数:7
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