An FKBP12 binding assay based upon biotinylated FKBP12

A binding assay was developed for measuring the affinity of FRBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP1 2 gene, and the fusion protein was expressed in Escherichia coli with bioti n ligase. The fusion protein was immobilized in avidin-coated multiwell pla tes, and varying concentrations of test ligands were allowed to compete wit h [H-3]FK506 for FKBP12 sites on the plate. The assay provided K-d values f or FK520, 32-hydroxy-ethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a conveni ent and rapid method for the assessment of FKBP12 binding by small molecule s. (C) 2001 Academic Press.