Discoverx Corporation

Keith R Olson

Thomas S Wehrman

William Raab

Wei Feng

Chin Yee Loh

Mahesh Mathrubutham

Philip Achacoso

Methods and genetic constructs are provided for detecting the binding of nuclear hormone receptors to a coactivator/corepressor. The methods employ enzyme fragment complementation using fragments of &#x3b2;-galactosidase as the detection system. Cells are transformed to express the large fragment of &#x3b2;-galactosidase fused to a member of the complex with NHR for initiation of transcription and have it localized in the nucleus and to express the small fragment of &#x3b2;-galactosidase fused to the nuclear hormone receptor for binding to the member upon stimulation with a ligand.

Assays for nuclear hormone receptor binding

Truncated fragments of the small fragment of &#x3b2;-galactosidase are provided that have low affinity for the large fragment of &#x3b2;-galactosidase and provide for robust signals when two fusion proteins are complexed due to the binding of the proteins to which the &#x3b2;-galactosidase fragments are fused. The truncated fragments do not interfere with the complexing of the two proteins and allow for the two proteins to function and be responsive to candidate compounds that affect complex formation.

&#x3b2;-galactosidase donor fragments

Methods for detecting phosphorylation of receptor tyrosine kinases ('RTKs') upon activation are provided. The method employs cells comprising two fusion products: (1) an RTK fused to a small fragment of &#x3b2;-galactosidase and (2) a phosphotyrosine binding peptide fused to the large fragment of &#x3b2;-galactosidase, where the 2 fragments weakly complex to form an active enzyme, and optionally a construct for a cytosolic RTK phosphorylating kinase, when the RTK does not autophosphoryate. To detect phosphorylation a &#x3b2;-galactosidase substrate is added to the cells, whereby product formation indicates the occurrence of phosphorylation.

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Pleasanton, CA, United States of America

Keith R Olson