Engineering The Glucocorticoid Receptor For Activation With Derivatives Of Fluorescent GFP Chromophores

                Nuclear Receptors are ligand activated transcription factors that play a critical role in the regulation of transcription. Currently, about 15% of drugs in the market target nuclear receptors and mutations in these receptors lead to diseases, ranging from cancer to diabetes. The human Glucocorticoid Receptor (GR) is a nuclear receptor that offers a lot of promise in drug design due to its unique properties in ligand binding and the various genes that it affects. This project involves the engineering of GR variants to bind a novel class of ligands. These ligands are derivatives of the chromophores found in the Green Fluorescent Protein (GFP). The variant proteins are created through random mutagenesis with an error prone PCR. The variants are then transformed into yeast and tested via chemical complementation. Chemical complementation is a selection system in yeast where upon binding of the small molecule to the receptor causes activation of an essential gene, in this case the HIS3 gene involved in histidine biosynthesis. The subsequent transformation of yeast Saccharomyces cerevisiae and selective plating resulted in successful colony generation. The colonies were grown on plates, with the chromophores present, to test for activation. Activation was confirmed with a restreak of each colony. A sequencing analysis which involves the comparison of the variant GR DNA with the Wild type is yet to be completed. Engineering variant GR protein will determine which amino acids are important to bind to chromophores as well as validate which functional groups on the chromophore are essential to binding.