Plishker M F, Rangwala S H, Berget P B
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston.
J Virol. 1988 Feb;62(2):400-6. doi: 10.1128/JVI.62.2.400-406.1988.
Two bacteriophage T4 proteins, P7 and P8, which are components of the phage baseplate have been purified to apparent homogeneity. P7 and P8 are the protein products of T4 genes 7 and 8. A plasmid has been constructed which contains approximately 5 kilobases of T4 DNA, including genes 7 and 8, under the control of the tac promoter. Induction of Escherichia coli W3110iQ cells containing this plasmid resulted in the production of functional P7 and P8. Standard protein isolation procedures were used to purify both P7 and P8 from extracts of induced cells. In T4-infected cells, these two proteins and P10 interact in a strictly ordered sequential manner (P10 + P7----P10/P7,P10/P7 + P8----P10/P7/P8) to form an intermediate in the baseplate assembly pathway. The three purified proteins assembled in vitro to form a limited number of oligomeric species, as determined by nondenaturing gel electrophoresis. P10 and P7 interacted in vitro to form two assemblies with distinct electrophoretic mobilities, both containing P10 and P7. Addition of P8 to this mixture resulted in the disappearance of both P10/P7 species and the appearance of a single new assembly with a different electrophoretic mobility. These interactions occurred without the addition of any catalyst or cofactors. Isolated P11 appeared to add as predicted to the in vitro-formed complexes without affecting the formation of the two P10/P7 or the single P10/P7/P8 intermediates. Interactions between P7 and P8 in the absence of P10 or interactions between P10 and P8 in the absence of P7 could not be detected. These data indicate that purified P10, P7, and P8 interact in vitro in a manner completely in accord with the published assembly pathway and thus establish a system for further study of the regulation of the formation of this assembly intermediate in vitro.
两种噬菌体T4蛋白，P7和P8，它们是噬菌体基板的组成成分，已被纯化至表观均一。P7和P8是T4基因7和8的蛋白质产物。构建了一种质粒，其在tac启动子的控制下包含约5千碱基的T4 DNA，包括基因7和8。诱导含有该质粒的大肠杆菌W3110iQ细胞可产生功能性的P7和P8。使用标准蛋白质分离程序从诱导细胞的提取物中纯化P7和P8。在T4感染的细胞中，这两种蛋白质与P10以严格有序的顺序相互作用（P10 + P7→P10/P7，P10/P7 + P8→P10/P7/P8），以在基板组装途径中形成中间体。通过非变性凝胶电泳测定，三种纯化的蛋白质在体外组装形成有限数量的寡聚体物种。P10和P7在体外相互作用形成两种具有不同电泳迁移率的组装体，两者均含有P10和P7。向该混合物中加入P8导致两种P10/P7物种消失，并出现一种具有不同电泳迁移率的单一新组装体。这些相互作用在不添加任何催化剂或辅因子的情况下发生。分离的P11似乎如预测的那样添加到体外形成的复合物中，而不影响两种P10/P7或单一P10/P7/P8中间体的形成。在没有P10的情况下P7和P8之间的相互作用或在没有P7的情况下P10和P8之间的相互作用无法检测到。这些数据表明，纯化的P10、P7和P8在体外以与已发表的组装途径完全一致的方式相互作用，从而建立了一个用于进一步研究体外这种组装中间体形成调控的系统。
