Komoda Y, Hijikata M, Tanji Y, Hirowatari Y, Mizushima H, Kimura K, Shimotohno K
Virology Division, National Cancer Center Research Institute, Tokyo, Japan.
Gene. 1994 Aug 5;145(2):221-6. doi: 10.1016/0378-1119(94)90009-4.
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.
丙型肝炎病毒（HCV）编码两种蛋白酶活性，即Cpro-1和Cpro-2。Cpro-1和Cpro-2似乎能加工它们所源自的前体多聚蛋白。含有这些蛋白酶区域的突变型HCV多肽在大肠杆菌中作为融合蛋白产生。HCV多肽的N端和C端分别与大肠杆菌麦芽糖结合蛋白（MBP）和大肠杆菌二氢叶酸还原酶（DHFR）融合。蛋白酶活性通过真核蛋白生产系统中使用的相同加工途径切割融合多肽。测定了加工后的融合蛋白的N端氨基酸（aa）序列。将这些N端序列与HCV前体多聚蛋白的aa序列进行比较，结果表明，HCV非结构（NS）蛋白之一p70（NS3）的N端和C端切割位点与其他加工研究中确定的位点相同：切割估计分别在HCV前体蛋白的aa 1026和1027之间以及aa 1657和1658之间，已知分别由Cpro-1和Cpro-2切割。Cpro-1和Cpro-2在大肠杆菌中均发挥作用，并具有真实的特征。
