Savilahti H, Caldentey J, Bamford D H
Department of Genetics, University of Helsinki, Finland.
Gene. 1989 Dec 21;85(1):45-51. doi: 10.1016/0378-1119(89)90462-9.
The gene VIII coding for the bacteriophage PRD1 terminal protein P8 has been cloned under the control of the lambda pL promoter. The recombinant plasmid thus obtained (pUSH20) was able to complement a mutation in the phage terminal-protein gene VIII. High expression of the cloned gene from this plasmid could be obtained by raising the growth temperature from 28 to 42 degrees C. This heat induction resulted in an increased synthesis of a protein of 30 kDa, the size expected for the P8 protein. When complemented with an extract of cells carrying the PRD1 DNA polymerase gene, the extract from the cells harboring the plasmid pUSH20 was able to form the P8-dGMP replication initiation complex. The PRD1 replication initiation reaction was optimized and used to detect the biological activity of the expressed terminal protein. Subsequently, P8 protein was purified to almost homogeneity and shown to be biologically functional after the various purification steps.
编码噬菌体PRD1末端蛋白P8的基因VIII已在λ pL启动子的控制下克隆。由此获得的重组质粒（pUSH20）能够弥补噬菌体末端蛋白基因VIII中的突变。通过将生长温度从28℃提高到42℃，可以从该质粒中获得克隆基因的高表达。这种热诱导导致一种30 kDa蛋白质的合成增加，这是P8蛋白预期的大小。当与携带PRD1 DNA聚合酶基因的细胞提取物互补时，含有质粒pUSH20的细胞提取物能够形成P8-dGMP复制起始复合物。对PRD1复制起始反应进行了优化，并用于检测表达的末端蛋白的生物学活性。随后，P8蛋白被纯化至几乎同质，并在各个纯化步骤后显示具有生物学功能。
