Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA.
Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA.
复制标题
大肠杆菌中从病毒 cDNA 合成的脊髓灰质炎病毒 RNA 聚合酶的酶活性。
DOI：
10.1016/0042-6822(88)90542-9
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发表时间：
1988
期刊：
影响因子：
3.7
通讯作者：
Ehrenfeld,E
中科院分区：
文献类型：
--
作者：
Rothstein,MA;Richards,OC;Amin,C;Ehrenfeld,E
已经构建了质粒，其含有指导脊髓灰质炎病毒RNA依赖性RNA聚合酶以重组融合蛋白形式表达的DNA序列。包含脊髓灰质炎病毒蛋白酶的额外基因导致融合蛋白裂解，产生52-kDa的酶活性聚合酶蛋白，与从病毒感染的HeLa细胞中分离的功能性酶明显相同。大量的聚合酶蛋白质在细菌中以颗粒或不溶性物质的形式积累，并且这种蛋白质几乎没有或没有活性。然而，回收了大量的可溶性活性酶，使得所得到的粗细菌提取物的比活性大于从病毒感染的HeLa细胞获得的比活性。从大肠杆菌中很容易地纯化出这种酶，并在寡聚（U）引物存在下产生一种将复制脊髓灰质炎病毒RNA作为模板的制备物。编码具有催化活性的RNA聚合酶的克隆DNA序列的可用性将允许遗传操作来启动这种酶的结构-功能研究。
Plasmids have been constructed that contain DNA sequences that direct the expression of the poliovirus RNA-dependent RNA polymerase, in the form of recombinant fusion proteins. Inclusion of an additional gene for the poliovirus protease results in cleavage of the fusion protein to yield a 52-kDa, enzymatically active, polymerase protein, apparently identical to the functional enzyme isolated from virus-infected HeLa cells. A large amount of polymerase protein accumulates as particulate or insoluble material in bacteria, and this protein has little or no activity. However, significant amounts of soluble, active enzyme are recovered, such that the resulting specific activity of crude bacterial extracts is greater than that obtained from virus-infected HeLa cells. Purification of the enzyme fromEscherichia coliis readily accomplished, and yields a preparation that will copy poliovirion RNA as template, in the presence of oligo(U) primer. The availability of cloned DNA sequences encoding catalytically active RNA polymerase will allow genetic manipulations to initiate structure-function studies of this enzyme.