Patent ID: 6117639
Filing Date: 2000-09-12
Classification: C07K,C12N,C12Q,G01N

Abstract:
A method for determining the inhibitory activity of a compound against a protease comprising the steps of:(A) in the absence of said compound, culturing in a first culture a host cell that expresses the protease to be assayed and a fusion protein, wherein said host cell comprises:(1) a first DNA molecule encoding the fusion protein or a vector comprising said first DNA molecule, wherein said fusion protein comprises:(a) a protease cleavage site(b) a ligand binding domain; and(c) a DNA binding domain;wherein the fusion protein also comprises an expression modulator domain, wherein said expression modulator domain regulates the transcription of a reporter gene; and wherein said protease cleavage site of said fusion protein is a target for cleavage by said protease; and(2) a second DNA molecule comprising:(a) a ligand-responsive element (""LRE""), which binds to said DNA binding domain of said fusion protein, wherein said binding is modulated by the presence of a ligand and is optionally modulated by the presence of a protein binding partner of said fusion protein;(b) a promoter which is modulated by said expression modulating domain of said fusion protein; and(c) a reporter gene, the expression of which is controlled by said promoter;(B) culturing in a second culture the host cell as used in step (A) in the presence of said compound under conditions which cause expression of said fusion protein;(C) adding to said first and to said second host cell cultures a ligand, wherein said ligand is required for said fusion protein to (i) bind to said LRE or to bind to a protein binding partner; and (ii) modulate the transcription of said reporter gene; and(D) comparing the amount of gene product produced from said reporter gene in said first host cell culture and said second host cell culture.