Mayer G, Launhardt H, Munder T
Hans-Knöll-Institut für Naturstoff-Forschung e.V. Jena, Germany.
Biotechniques. 1999 Jul;27(1):86-8, 92-4. doi: 10.2144/99271st01.
The two-hybrid system in Saccharomyces cerevisiae is a genetic approach for the detection of of protein-protein interactions in vivo. This technology relies on the the activity of separated DNA-binding and transactivation domains of specific transcription factors to reconstitute an active transcription factor complex if interacting proteins are fused to these domains. Interactions are consequently detected through the activity of reporter genes. The two-hybrid technology has been successfully applied for the determination of interactions between numerous proteins of several organisms. Conventional reporter systems, such as the beta-galacatosidase from Escherichia coli, suffer from a variety of drawbacks, including the requirement for external substrates. In this report, we describe an alternative version of the two hybrid system using the combined advantages of the copper-inducible transcription factor Acel together with the yeast metallothionein gene CUP1 and the green fluorescence protein from aquatic invertebrates as reporters. This technique allows the copper-dependent monitoring of protein-protein interactions in living yeast cells.
酿酒酵母中的双杂交系统是一种用于在体内检测蛋白质-蛋白质相互作用的遗传学方法。该技术依赖于特定转录因子分离的DNA结合域和反式激活域的活性，以便在相互作用的蛋白质与这些结构域融合时重建活性转录因子复合物。因此，通过报告基因的活性来检测相互作用。双杂交技术已成功应用于多种生物体中众多蛋白质之间相互作用的测定。传统的报告系统，如来自大肠杆菌的β-半乳糖苷酶，存在多种缺点，包括需要外部底物。在本报告中，我们描述了双杂交系统的一种替代形式，它利用了铜诱导转录因子Acel与酵母金属硫蛋白基因CUP1以及来自水生无脊椎动物的绿色荧光蛋白作为报告基因的综合优势。该技术允许对活酵母细胞中的蛋白质-蛋白质相互作用进行铜依赖性监测。
