Patent ID: 6303310
Filing Date: 2001-10-16
Classification: C12N,C12Q

Abstract:
A method for screening a library for peptides or proteins that will facilitate multiple protein interactions by detecting multiple protein interactions, the method comprising:(a) providing a yeast or mammalian host cell which is deficient in at least a first selectable marker, a second selectable marker, a third selectable marker, and a fourth selectable marker, said host cell comprising a detectable gene wherein the detectable gene expresses a detectable protein when the detectable gene is activated by an amino acid sequence including a transcriptional activation domain when the transcriptional activation domain is in sufficient proximity to the detectable gene, said detectable protein being said fourth selectable marker;(b) providing a first plasmid comprising said first selectable marker and a first chimeric gene under control of a constitutive promoter that is expressed in the host cell, the first chimeric gene comprising a DNA sequence that encodes a first hybrid protein, the first hybrid protein comprising:(i) a DNA-binding domain that recognizes a binding site on the detectable gene in the host cell; and(ii) a first test protein or fragment thereof that is to be tested for interaction with a second test protein or fragment thereof and at least one mediating test protein or fragment thereof, wherein said first test protein or fragment thereof and said second test protein or fragment thereof do not interact directly or interact only through mediation by a mediating test protein or fragment thereof;(c) providing a second plasmid comprising said second selectable marker and a second chimeric gene under control of a constitutive promoter that is expressed in the host cell, the second chimeric gene comprising a DNA sequence that encodes a second hybrid protein, the second hybrid protein comprising:(i) the transcriptional activation domain; and(ii) the second test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and with at least one mediating test protein or fragment thereof;(d) providing a library of third plasmids comprising said third selectable marker and at least one mediating chimeric gene under control of a constitutive promoter that is expressed in the nucleus of the host cell, each mediating chimeric gene comprising a DNA sequence that encodes a mediating hybrid cytoplasmic protein, the mediating hybrid protein comprising:(i) a nuclear localization peptide; and(ii) a mediating test protein or fragment thereof that is to be tested for interaction with the first test protein or fragment thereof and the second test protein or fragment thereof;wherein interaction between the first test protein or fragment thereof, the second test protein or fragment thereof, and the mediating test protein or fragment thereof in the host cell causes the transcriptional activation domain to activate transcription of the detectable gene;(e) introducing the first plasmid, the second plasmid, and a third plasmid into the host cell;(f) subjecting the host cell to conditions under which the first hybrid protein, the second hybrid protein, and the mediating hybrid protein are expressed, each under the control of their constitutive promoter, in sufficient quantity for the detectable gene to be activated; and,(g) detecting multiple protein interactions by determining whether the detectable gene has been expressed to a degree greater than expression in a control group of yeast or mammalian host cells which do not contain said first chimeric gene, said second chimeric gene and said mediating chimeric gene.