Protocol

Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array: Amplification of ORFs

This protocol was adapted from "Genome-Wide Analysis of Protein-Protein Interactions Using a Two-Hybrid Array," contributed by Tony R. Hazbun and John P. Miller, Chapter 37, in Protein-Protein Interactions, 2nd edition (eds. Golemis and Adams). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

INTRODUCTION

The two-hybrid array method described in this protocol was applied to ~6000 open reading frames (ORFs) of the Saccharomyces cerevisiae genome, but it could be applied to any collection of ORFs, including the genome complements of other organisms or smaller sets of functionally related ORFs. The method relies on a spatially ordered set of yeast strains, each expressing a different protein fused to the Gal4 activation domain (AD). A mating strategy is used in which a strain expressing a protein fused to the Gal4 DNA-binding domain (BD) (the bait) is mated to each array strain (the prey). Construction of the array is achieved using polymerase chain reaction (PCR) and recombinational cloning in conjunction with high-throughput transformation of the appropriate yeast strain. To capitalize on the advantageous use of the mating procedure, the array strains must be haploid yeast. This allows the introduction of the Gal4 BD and AD fusions into the same diploid cell by mating of the haploid array strains with a haploid strain of the opposite mating type containing the BD. Diploid yeast strains expressing both of these protein fusions are selected using auxotrophic media, and subsequently, two-hybrid positives are selected for the activation of a reporter gene that allows their growth on the appropriate media. This protocol describes the first step in constructing an array: amplification of the predicted ORFs that are to be included in the array. Gene-specific primers containing vector-specific flanking sequences that facilitate recombinational cloning are used to amplify each ORF. A secondary amplification can be used to extend the length of the homologous vector sequence flanking the ORF.