Specific Affinity Extraction Method for Small Molecule-Binding Proteins

Abstract

Validation of the targets of candidate drugs is critical for rapid and efficient drug discovery and development and for understanding the pharmacological action and potential toxicities of the prospective therapeutic agent. Due to the nonspecific binding of abundant proteins to small molecule-immobilized gels, it is difficult to identify the protein targets of small molecules from crude biological samples by affinity extraction. To address this problem, we have developed an affinity gel for the specific extraction of small molecule-binding proteins. We immobilized small molecules on the agarose gel through a disulfide linker that is cleavable by mild reduction. This system has allowed specific and noncovalent complex formation between the small molecule and the target protein, keeping the effect of the nonspecific abundant proteins adsorbed on both the linker and gel surface to minimum. By preparing this affinity matrix with deoxycholate as a model small molecule, we captured two independent deoxycholate-binding proteins of different affinities from mouse ascites, anti-deoxycholate antibody, and serum albumin. As other proteins were not captured, this affinity extraction method should contribute significantly to the accurate and rapid drug discovery and development.