Fuller K J, Morse M A, White J H, Dowell S J, Sims M J
Immunology Unit, Glaxo Wellcome Medicines Research Centre, Stevenage, Hertfordshire, UK.
Biotechniques. 1998 Jul;25(1):85-8, 90-2. doi: 10.2144/98251st04.
We describe a yeast trihybrid system that facilitates rapid screening of cDNA libraries. Novel yeast vectors were developed that direct integration of cDNA encoding the bait and third protein component into the yeast chromosome. A recombinant yeast strain is thus generated (screening strain) and is available for library transformation. Transformation with the library DNA is a single, efficient transformation event, allowing the cDNA library to be represented in one step. Recovery of the library plasmid from the yeast is also simplified, since it is the only episomal plasmid. Assay of trihybrid interaction and identification of positive clones is facilitated by regulating expression of the third protein component using the yeast MET3 promoter, which is repressed in the presence of exogenous methionine. Trihybrid interactions are detected only on media lacking methionine. This trihybrid system uses the standard E. coli LacZ and yeast HIS3 reporter genes and is compatible with most available Gal4 activation domain cDNA libraries. We describe the successful application of this yeast trihybrid system to the study of phosphoprotein interactions involved in T-cell signaling.
我们描述了一种有助于快速筛选cDNA文库的酵母三杂交系统。开发了新型酵母载体，可将编码诱饵蛋白和第三种蛋白质组分的cDNA定向整合到酵母染色体中。由此产生一种重组酵母菌株（筛选菌株），可用于文库转化。用文库DNA进行转化是一个单一、高效的转化事件，能使cDNA文库一步到位得以呈现。从酵母中回收文库质粒也得以简化，因为它是唯一的附加体质粒。使用酵母MET3启动子调节第三种蛋白质组分的表达，有助于三杂交相互作用的检测和阳性克隆的鉴定，该启动子在存在外源甲硫氨酸时受到抑制。仅在缺乏甲硫氨酸的培养基上才能检测到三杂交相互作用。这种三杂交系统使用标准的大肠杆菌LacZ和酵母HIS3报告基因，并且与大多数现有的Gal4激活结构域cDNA文库兼容。我们描述了这种酵母三杂交系统在研究参与T细胞信号传导的磷蛋白相互作用中的成功应用。
