Takahara Y, Ando N, Kohara M, Hagino-Yamagishi K, Nomoto A, Itoh H, Numao N, Kondo K
Genetic Engineering Section, Sagami Chemical Research Center, Kanagawa, Japan.
Gene. 1989 Jul 15;79(2):249-58. doi: 10.1016/0378-1119(89)90207-2.
A viral protein 3C of the poliovirus (PV) Sabin 2 strain, a possible core region of the viral proteinase, was expressed in Escherichia coli using a recombinant DNA technology. The protein was recovered as a soluble protein from the insoluble protein fraction of the bacterial lysate, and was purified by a simple procedure with column chromatography. The viral capsid precursor P1 (1ABCD) of the PV Sabin 3 strain, which had been similarly produced in E. coli, was mixed with the purified or crude recombinant 3C. Immunoblotting assay with monoclonal antibodies specific to capsid proteins 1C (VP3) and 1D (VP1) of the PV Sabin 3 strain revealed that the in vitro reaction products contained 1C (VP3), 1D (VP1) and 1ABC (VP0-VP3). The data indicated that processing of the polyprotein P1 by the recombinant 3C proceeded properly in vitro, although an undigested product, 1ABC, is always detected in the reaction mixture. The results strongly suggest that, in addition to a protein 3CD, the 3C protein itself is also catalytically active in the processing of the viral capsid precursor polyprotein P1.
利用重组DNA技术在大肠杆菌中表达了脊髓灰质炎病毒（PV）萨宾2型毒株的一种病毒蛋白3C，它可能是病毒蛋白酶的核心区域。该蛋白从细菌裂解物的不溶性蛋白部分中作为可溶性蛋白回收，并通过柱层析的简单程序进行纯化。同样在大肠杆菌中产生的PV萨宾3型毒株的病毒衣壳前体P1（1ABCD）与纯化的或粗制的重组3C混合。用针对PV萨宾3型毒株衣壳蛋白1C（VP3）和1D（VP1）的单克隆抗体进行免疫印迹分析表明，体外反应产物含有1C（VP3）、1D（VP1）和1ABC（VP0-VP3）。数据表明，尽管在反应混合物中总是检测到未消化的产物1ABC，但重组3C对多蛋白P1的加工在体外进行得很顺利。结果强烈表明，除了蛋白3CD外，3C蛋白本身在病毒衣壳前体多蛋白P1的加工中也具有催化活性。
