Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator.
Two-hybrid Systems Stage 2: Selecting an Interactor Protocol In this stage of the protocol, a mammalian cDNA library constructed in a plasmid such as pJG4-5 is transformed into yeast strains containing pBAIT and the lexAop-lacZ reporter plasmid. PJG4-5 expresses the cloned cDNAs from a cassette containing a transcriptional activation domain and other moieties under the control of the yeast GAL1 promoter.