Mendelsohn Andrew R
The Molecular Sciences Institute, Berkeley, California, USA.
Curr Protoc Mol Biol. 2004 Feb;Chapter 20:Unit 20.8. doi: 10.1002/0471142727.mb2008s65.
Protein-protein interactions play a critical role in biology. Disrupting a specific protein-protein interaction and studying the resulting phenotype can help elucidate the function of the interaction. A method to rapidly make and identify mutant proteins that no longer bind to a specific partner protein is described. The method takes advantage of the ease of the interaction trap/two-hybrid system and PCR mutagenesis. PCR fusion of the target protein to GFP is used to ensure the protein's open reading frame is not disrupted by mutagenesis. The resulting noninteracting mutant proteins usually result from a single missense mutation, which can easily be identified.
蛋白质-蛋白质相互作用在生物学中起着关键作用。破坏特定的蛋白质-蛋白质相互作用并研究由此产生的表型有助于阐明这种相互作用的功能。本文描述了一种快速制备和鉴定不再与特定伴侣蛋白结合的突变蛋白的方法。该方法利用了相互作用陷阱/双杂交系统的简便性和PCR诱变技术。将目标蛋白与绿色荧光蛋白（GFP）进行PCR融合，以确保蛋白质的开放阅读框不会因诱变而被破坏。产生的非相互作用突变蛋白通常源于单个错义突变，这种突变很容易被识别。
