Hidaka M, Yoshida Y, Masaki H, Uozumi T
Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
Biosci Biotechnol Biochem. 1993 May;57(5):710-4. doi: 10.1271/bbb.57.710.
We have cloned a 5-kb cDNA corresponding to the 3'-half of genomic RNA of potato virus Y (PVY), the type member of plant potyvirus. Based on its nucleotide sequence, the cDNA was presumed to encode PVY protease responsible for the self-processing of polyprotein precursor expressed from PVY genome. To test its proteolytic activity, the cDNA was modified so as to express the protease-polymerase-coat protein (CP) portion of PVY polyprotein in Escherichia coli. By Western blotting analysis of the cell extract of E. coli expressing the polyprotein, mature CP was detected. A large deletion in the polymerase-coding region did not affect the emergence of mature CP, but a small deletion in the putative protease region resulted in disappearance of mature CP and appearance of the intact polyprotein. These results indicated that the polyprotein expressed in E. coli was cleaved depending on the proteolytic activity of PVY protease.
我们克隆了一个5kb的cDNA，它对应于植物马铃薯Y病毒（PVY）基因组RNA的3'端一半，PVY是植物马铃薯Y病毒属的典型成员。根据其核苷酸序列，推测该cDNA编码负责从PVY基因组表达的多聚蛋白前体自我加工的PVY蛋白酶。为了测试其蛋白水解活性，对该cDNA进行了修饰，以便在大肠杆菌中表达PVY多聚蛋白的蛋白酶-聚合酶-衣壳蛋白（CP）部分。通过对表达多聚蛋白的大肠杆菌细胞提取物进行蛋白质免疫印迹分析，检测到了成熟的CP。聚合酶编码区的大片段缺失并不影响成熟CP的出现，但假定蛋白酶区的小片段缺失导致成熟CP消失，完整多聚蛋白出现。这些结果表明，在大肠杆菌中表达的多聚蛋白是根据PVY蛋白酶的蛋白水解活性进行切割的。
