Yeast Two-Hybrid Library Screening
The two-hybrid system was originally devised by Fields and Song as a protein interaction detection system in yeast (1). Subsequently, it has been employed in many laboratories as a means of screening cDNA and genomic fusion libraries for protein interaction partners (2, 3, 4, 5, 6, 7, 8). The method relies on the fact that transcription factors such as the yeast GAL4 factor consist of separable DNA-binding and transcriptional regulatory domains, the former being required to direct the latter to appropriate promoters where transcriptional activation is effected, usually by direct or indirect interaction of the activation domain with the general transcription machinery. The essence of the two-hybrid system is the in vivo reconstitution of a functional transcriptional activator from two interacting polypeptides, one fused to a sequence-specific DNA binding domain and the other to a potent transcriptional activation domain that is detected by the activation of a reporter gene or genes (see Fig. 1). For the purposes of identifying new protein partners, DNA encoding the polypeptide for which partners are sought (bait polypeptide) is ligated into a yeast shuttle vector to create a fusion protein with the DNA binding domain of GAL4 (see Fig. 2 A) or sometimes the bacterial LexA DNA binding protein (6, 7, 8, 9, 10). Library cDNA is ligated into a second shuttle vector to create an activation domain-tagged cDNA library (see Fig. 2 B). In the original method as proposed by Chien et al. (2), the bait (GAL4 DNA binding domain) construct and activation domain fusion library were cotransformed into a yeast strain containing an integrated lacZ gene driven by a GAL4-responsive promoter (2).
KeywordsActivation Domain Bait Plasmid Sterile Toothpick Adenine Sulphate Bait Construct
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