Patent ID: 7479377
Filing Date: 2009-01-20
Classification: C07K,C12N,C12Q,C40B,G01N

Abstract:
1. A method for determining a change in degradation of a cellular protein in a cellular pathway in response to a candidate compound, said method employing a fragment of from 37 to 110 amino acids of β-galactosidase as an enzyme donor (“ED”) and a larger fragment of said β-galactosidase as an enzyme acceptor (“EA”), wherein the two fragments are characterized by complexing to form an active enzyme, further said method employing a mammalian cell into which is introduced a genetic expression construct encoding a surrogate fusion protein comprising said ED and a surrogate for said cellular protein, said method comprising: expressing said fusion protein in said cell in the presence of said candidate compound, which fusion protein serves as a surrogate for said cellular protein in response to said candidate compound; lysing said cell to form a lysate; providing to said lysate a purified EA and a substrate for the ED/EA active complex wherein the complex converts the substrate into a detectable product; and measuring the level of said detectable product produced by the method with the cell in the presence of said candidate compound in comparison to the level of said detectable product produced by the method with a cell in the absence of said candidate compound, wherein a decrease in level of said detectable product in the presence of said candidate compound in comparison to the level of said detectable product in the absence of said candidate compound is indicative of said degradation of said cellular protein in response to said candidate compound.