Journal
BIOCHEMICAL PHARMACOLOGY
Volume 63, Issue 9, Pages 1619-1628Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/S0006-2952(02)00884-5
Keywords
yeast three-hybrid; GAL4; dexamethasone; FK506; methotrexate; glucocorticoid receptor
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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 106 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. (C) 2002 Published by Elsevier Science Inc.
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