Brown W C, Campbell J L
Braun Laboratories, Division of Chemistry, California Institute of Technology, Pasadena 91125.
Gene. 1993 May 15;127(1):99-103. doi: 10.1016/0378-1119(93)90622-a.
Here, we describe a modification of a plasmid, pT7-7 [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 262 (1985) 1074-1078], that allows expression of inserted genes from the phage T7 RNA polymerase promoter. The modification is designed to suppress readthrough transcription from cryptic promoters and start points on the plasmid, in order to reduce expression in the absence of T7 RNA polymerase and thus improve the vector for use in the expression of highly toxic gene products. This vector (pT7SC) was used to stably clone the POL3 gene (encoding DNA polymerase delta) of Saccharomyces cerevisiae, which destabilizes all other cloning and expression vectors tested. Previously described expression strategies proved ineffective in overexpressing the POL3 gene. A new strategy was developed which relies on induction by infection with mutant T7 phage. This system efficiently overproduced the POL3 gene product.
在此，我们描述了对质粒pT7 - 7 [塔博尔和理查森，《美国国家科学院院刊》262 (1985) 1074 - 1078] 的一种修饰，该修饰可使 T7 噬菌体 RNA 聚合酶启动子驱动插入基因的表达。这种修饰旨在抑制来自质粒上隐蔽启动子和起始位点的通读转录，以减少在没有 T7 RNA 聚合酶时的表达，从而改进该载体用于表达高毒性基因产物。此载体（pT7SC）用于稳定克隆酿酒酵母的 POL3 基因（编码 DNA 聚合酶δ），该基因会使所有其他测试的克隆和表达载体不稳定。先前描述的表达策略在过表达 POL3 基因方面证明无效。开发了一种新策略，该策略依赖于用突变 T7 噬菌体感染进行诱导。该系统高效过量产生 POL3 基因产物。
