Method of detecting biologically active substances by using green fluorescent protein
AssigneeNovo Nordisk A/S
InventorsOle Thastrup, S.o slashed.ren Tullin, Lars Kongsbak Poulsen, Sara Petersen Bj.o slashed.rn
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Abstract
The present invention provides a method for detecting a biologically active substance that affects intracellular processes mediated through a protein kinase or second messenger, which comprises incubating a cell with a test substance and measuring any change caused by the test substance in the fluorescence of (i) green fluorescent protein (GFP) having a protein kinase recognition site or (ii) GFP that has been modified to contain a second messenger binding domain.
Claims23
A method of detecting a substance affecting intracellular processes mediated by a protein kinase, said method comprising
- (a) culturing a cell expressing green fluorescent protein (GFP) having a protein kinase recognition site, wherein fluorescence of said GFP changes upon phosphorylation at said protein kinase recognition site;
- (b) measuring the fluorescence of said GFP in said cell in the absence and presence of a test sample;
- and
- (c) comparing the GFP fluorescences measured in step (b);
- wherein a difference between the fluorescence measured in the absence and presence of said sample indicates the presence in said sample of a substance that affects intracellular processes mediated by a protein kinase.
A method of detecting a substance affecting intracellular processes mediated by a second messenger, said method comprising
- (a) culturing a cell expressing a modified GFP comprising a binding domain of a second messenger, wherein fluorescence of said modified GFP changes upon binding of said second messenger to said binding domain;
- (b) measuring the fluorescence of said modified GFP in said cell in the absence and presence of a test sample;
- and
- (c) comparing the GFP fluorescences measured in step (b);
- wherein a difference between the fluorescence measured in the absence and presence of said sample indicates the presence in said sample of a substance that affects intracellular processes mediated by a second messenger.
An isolated DNA encoding a modified GFP wherein one or more amino acids have been substituted, inserted, or deleted to provide a protein kinase recognition site, wherein fluorescence of said GFP changes upon phosphorylation at said protein kinase recognition site.
An isolated DNA encoding a modified GFP comprising a binding domain of a second messenger, wherein fluorescence of said modified GFP changes upon binding of said second messenger to said binding domain.
Dependencies are inferred from the claim text.