Patent ID: 6171792
Filing Date: 2001-01-09
Classification: C07K,C12N,C12Q,G01N

Abstract:
A method for detecting a protein--protein interaction, comprising:(a) providing a host cell which contains(i) a first reporter gene operably linked to a DNA sequence comprising a first protein binding site;(ii) a second reporter gene operably linked to a DNA sequence comprising a second protein binding site;(iii) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising a first protein covalently bonded to a binding moiety which is capable of specifically binding to said first protein binding site;(iv) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising a second protein covalently bonded to a binding moiety which is capable of specifically binding to said second protein binding site; and(v) a third fusion gene which expresses a third fusion protein, said third fusion protein comprising a third protein covalently bonded to a gene activating moiety;(b) measuring expression output of said first reporter gene as a measure of said interaction between said first and said third proteins;(c) measuring expression output of said second reporter gene as a measure of said interaction between said second and said third proteins;(d) interpreting the expression output results of step (b) and step (c), whereby(i) increased output of both of said first and said second reporter genes indicates that said third fusion protein interacts with both of said first and said second fusion proteins;(ii) increased output of said first reporter gene but not said second reporter gene indicates that said third fusion protein interacts with said first fusion protein but not said second fusion protein;(iii) increased output of said second reporter gene but not said first reporter gene indicates that said third fusion protein interacts with said second fusion protein but not said first fusion protein; and(iv) no change in output in either of said first or said second reporter genes indicates that said third fusion protein does not interact with either of said first or said second fusion genes;(e) comparing said expression output results of step (b) and step (c) with the expression output result measured in either(i) a first comparison host cell which contains(a) a reporter gene operably linked to a DNA sequence comprising a protein binding site;(b) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said first protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and(c) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety; or(ii) a second comparison host cell which contains(a) a reporter gene operably linked to a DNA sequence comprising a protein binding site;(b) a first fusion gene which expresses a first fusion protein, said first fusion protein comprising said second protein covalently bonded to a binding moiety which is capable of specifically binding to said protein binding site; and(c) a second fusion gene which expresses a second fusion protein, said second fusion protein comprising said third protein covalently bonded to a gene activating moiety; or(iii) both of said first and said second comparison host cells.