Patent ID: 6187535
Filing Date: 2001-02-13
Classification: C12N,C12Q

Abstract:
A method for selecting a polynucleotide encoding a prey polypeptide, said prey polypeptide being able to interact with a bait polypeptide, comprising the steps of:(A) providing a yeast cell mixture comprising a first population of yeast cells containing at least one first haploid recombinant yeast cell containing a polynucleotide encoding said bait polypeptide (bait polynucleotide) and a second population of yeast cells containing at least one second haploid recombinant yeast cell containing a polynucleotide encoding said prey polypeptide (prey polynucleotide);(B) subjecting the polynucleotide encoding the bait polypeptide to a two-hybrid screening method, wherein said two-hybrid screening method comprises a step of mating for a maximum of 5 hours, without replica plating, said at least one first haploid recombinant yeast cell containing the bait polynucleotide with said second haploid recombinant yeast cell containing the prey polynucleotide, provided that one haploid yeast cell among the first haploid recombinant yeast cell or the second haploid recombinant yeast cell also contains at least one detectable gene that is activated by a polypeptide including a transcriptional activation domain;(C) selecting the recombinant diploid yeast cell obtained at step (B) for which the detectable gene has been expressed to a degree greater than expression in the absence of interaction between the bait polypeptide and the prey polypeptide; and(D) optionally characterizing the prey polynucleotide contained in each diploid yeast cell selected at step (C).