The interaction trap: In vivo analysis of protein-protein associations

This chapter describes several methods used in characterizing interactions between the products of previously cloned genes and to construct and screen an Arabidopsis library using the interaction trap. It describes in detail several of the methods used to generate bait and prey fusions with previously cloned Arabidopsis sequences and for the construction of an Arabidopsis expression library in the prey vector. Methods for testing baits in repression and activation assays, for high-efficiency transformation of plasmids into yeast, and for characterizing the positive clones isolated in library screens are described. The first step in the use of the interaction trap for the analysis of interactions between two known proteins or for screening an expression library with a single known protein is to construct a bait fusion. All of the plasmids used in the interaction trap are shuttle vectors that confer ampicillin resistance in Escherichia coli and uracil (URA), tryptophan (TRP), or histidine (HIS) prototrophy in the yeast strains EGY40 and EGY48. A basic requirement of the interaction assay is that the LexA fusion protein, the bait, causes little or no transcriptional activation of reporter genes in the absence of the activation fusion, the prey. © 1995, Academic Press Inc.