柯萨奇病毒B3重组3C蛋白酶的表达与纯化

Expression and purification of recombinant 3C proteinase of Coxsackievirus B3.

作者信息

Miyashita K, Kusumi M, Utsumi R, Komano T, Satoh N

机构信息

Central Research Laboratories, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan.

出版信息

Biosci Biotechnol Biochem. 1992 May;56(5):746-50. doi: 10.1271/bbb.56.746.

DOI:10.1271/bbb.56.746

PMID:1369382

Abstract

We have cloned various lengths of coxsackievirus B3 cDNA encompassing the region encoding the 3C proteinase, which is essential to the viral replication cycle. Such viral cDNAs were fused in frame to the 5'terminal portion of the lacZ' gene carried on the vector pUC118 to express mature 3C proteinase in Escherichia coli. In the E. coli cells containing pCXB108 or pCXB117, constructed for this study, a large amount of 23-kDa protein was synthesized in the presence of IPTG. This protein was purified and was shown to be intact 3C proteinase. These data suggest that 3C proteinase, expressed as a part of a fusion protein, was active in E. coli and released itself from the precursor fusion protein by autocatalytic cleavage.

摘要

我们克隆了柯萨奇病毒B3不同长度的cDNA，这些cDNA包含编码3C蛋白酶的区域，该蛋白酶对病毒复制周期至关重要。此类病毒cDNA与载体pUC118上携带的lacZ'基因的5'末端部分进行读码框融合，以便在大肠杆菌中表达成熟的3C蛋白酶。在本研究构建的含有pCXB108或pCXB117的大肠杆菌细胞中，在异丙基-β-D-硫代半乳糖苷（IPTG）存在的情况下合成了大量23 kDa的蛋白质。该蛋白质经纯化后显示为完整的3C蛋白酶。这些数据表明，作为融合蛋白一部分表达的3C蛋白酶在大肠杆菌中具有活性，并通过自催化切割从前体融合蛋白中释放出来。