アフィニティービーズによるケミカルバイオロジーへの挑戦

抄録

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 the identification of target proteins.
This study describes the preparation and use of affinity beads for the identification of drug receptors. The drugs of interest are immobilized onto a matrix that consists of latex beads covalently coupled with spacers. The latex beads are composed of glycidylmethacrylate (GMA)-covered GMA-styrene (St) copolymer cores (SG beads), which were originally developed for the affinity purification of DNA-binding proteins. To reduce steric hindrance, a divalent epoxide, ethyleneglycol diglycidylether (EGDE) molecules are introduced into the SG beads (SGE beads) as spacers after aminolysis of the epoxy groups on the surfaces of the beads.
The new SGE beads offer several distinct advantages compared to 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 covalently coupled to the SGE beads. A typical target protein is purified more than 1000 fold, with a yield of more than 70%. If necessary, the batch purification step can be repeated, further increasing the purity of the target protein. This procedure is not only effective, but is also simple and straightforward to perform. Using the affinity beads, we have identified multiple drugs receptors, which can then be used in such applications as drug screening, drug design, and evaluation of drug efficiency for given individuals.