A GAL4-based yeast three-hybrid system for the identification of small molecule-target protein interactions.
We report the development of a yeast strain designed for assaying compound-protein interactions through activation of reporter gene expression. Activation of lacZ expression, driven by the GAL4 promoter, has been demonstrated for precedented compound-protein interactions between FK506 and FK506 binding protein 12 (FKBP12) and also between methotrexate (MTX) and dihydrofolate reductase (DHFR). Reporter gene expression was completely abrogated in a competitive manner by the presence of excess FK506 or MTX, respectively. In addition, a strain expressing a mutated DHFR clone with decreased binding affinity for MTX was not capable of activating reporter gene expression. While strain sensitivity is compound-dependent, the minimum compound concentration necessary to drive reporter gene expression was 20 nM for the FK506-FKBP12 interaction. The utility of this strain as a tool for identifying unknown compound-binding proteins has been demonstrated by screening a mouse cDNA library for clones that encode proteins capable of binding MTX. Four library clones of mouse DHFR were identified after screening 5 x 10(6) clones. The screen background was low and false positives were easily identified, making this yeast system particularly amenable for use in a screening context for novel compound-protein interactions.
Henthorn
Debbie C
DC
Asthma Cell Biology, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Hertfordshire, Stevenage, UK. dch47508@gsk.com
Jaxa-Chamiec
Albert A
AA
Meldrum
Eric
E
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D001709:Biotechnology / Q000379:methods*; D004268:DNA-Binding Proteins; D003907:Dexamethasone / Q000737:chemistry / Q000494:pharmacology; D019281:Dimerization; D005656:Fungal Proteins / Q000235:genetics; D017930:Genes, Reporter / Q000187:drug effects; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D008727:Methotrexate / Q000737:chemistry / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D016559:Tacrolimus / Q000494:pharmacology; D022061:Tacrolimus Binding Protein 1A / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation; D015003:Yeasts / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D007166:Immunosuppressive Agents; D011965:Receptors, Glucocorticoid; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D003907:Dexamethasone; D000429:Alcohol Oxidoreductases; C056570:dihydroflavanol 4-reductase; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus; D008727:Methotrexate