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New approach to chemical biology for drug discovery: construction of new affinity beads and their application
Arthritis Res Thervolume 5, Article number: 85 (2003)
Affinity purification is an established technique used to identify ligand-binding proteins; however, its widespread use has been limited by the inefficiency and instability of conventional matrices. The unstable nature of conventional matrices narrows the spectrum of ligands that can be used. Moreover, nonspecific binding of proteins to the solid support has complicated identification of target proteins. Another difficulty is frequent low purification efficiency, which requires partial purification of the source material prior to affinity chromatography to improve results.
The present article describes the preparation and use of affinity beads for identification of drug receptors. The drugs of interest are immobilized to the matrix, which consists of latex beads covalently coupled with spacers. The latex beads are composed of glycidylmethacrylate-covered glycidylmethacrylate–styrene copolymer cores (SG beads) that were developed originally for the affinity purification of DNA-binding proteins. To reduce steric hindrance, divalent epoxide, ethyleneglycol diglycidylether molecules are introduced to the SG beads (SGE beads) as spacers after aminolysis of epoxy groups on the surfaces of the beads.
The new SGE beads offer several distinct advantages compared with commonly used supports such as agarose beads. These advantages have enabled us to identify drug receptors directly from crude cell extracts within a few hours. Drug derivatives with a reactive group are then covalently coupled to the SGE beads. Crude cell extracts are incubated with the drug affinity beads. The desired drug-binding proteins are then purified in a batchwise manner. The desired drug-binding proteins bind to the drug on the affinity beads, while other proteins flow through the beads. The drug-binding proteins are eluted from the affinity beads with a high salt solution or a detergent containing solution following brief centrifugation. A typical target protein is purified more than 1000-fold with a yield of 70%. If necessary, the batchwise step can be repeated, enabling further purification. This procedure is not only effective, but is also simple and straightforward to perform. Using the affinity beads, we have identified multiple drug receptors, which could be utilized for drug screening, drug design, evaluation of drug efficiency for a given individual, and so on.