Cheng X, Patterson T A
DuPont Merck Pharmaceutical Company, Wilmington, DE 19880-0400.
Nucleic Acids Res. 1992 Sep 11;20(17):4591-8. doi: 10.1093/nar/20.17.4591.
A set of plasmid vectors which allow single-step cloning and expression of PCR-amplified DNA coding sequences has been constructed. The vectors contain the phage lambda PL promoter, a synthetic translation initiation region (TIR), and convenient cloning sites. The cloning sites provide all or part of an AUG translation initiation codon and facilitate the precise fusion of target DNA sequences to vector transcriptional and translational signals. The vectors were constructed with synthetic TIRs because there is evidence which suggests that the efficiency of the phage lambda cII gene TIR present in the parental vector depends strongly on information contained within the cII N-terminal coding sequence. Bovine brain 14-3-3 eta chain cDNA was PCR-amplified and used to demonstrate the expression capacity of the newly constructed vectors. A significant increase in expression of 14-3-3 protein was observed when synthetic TIRs were used in the place of the cII TIR. Expression levels vary from 15% to 48% of total cell protein. The effects of a reported translational enhancer from phage T7 on expression of the 14-3-3 protein are also discussed. The vectors should be generally useful for high level heterologous protein expression in Escherichia coli.
已经构建了一组质粒载体，可用于PCR扩增的DNA编码序列的单步克隆和表达。这些载体包含噬菌体λ PL启动子、一个合成翻译起始区域（TIR）和方便的克隆位点。这些克隆位点提供了全部或部分AUG翻译起始密码子，并有助于将目标DNA序列精确融合到载体转录和翻译信号上。构建这些载体时使用了合成TIR，因为有证据表明亲本载体中存在的噬菌体λ cII基因TIR的效率在很大程度上取决于cII N端编码序列中包含的信息。牛脑14-3-3 η链cDNA经PCR扩增后用于证明新构建载体的表达能力。当使用合成TIR替代cII TIR时，观察到14-3-3蛋白的表达显著增加。表达水平占总细胞蛋白的15%至48%。还讨论了噬菌体T7报道的翻译增强子对14-3-3蛋白表达的影响。这些载体通常应可用于在大肠杆菌中高水平表达异源蛋白。
