title: A new efficient method of generating photoaffinity beads for drug target identification.
abstract: Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments.
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan. Electronic address: yoichi.nishiya@kyowa-kirin.co.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nishiya
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Hamada
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Abe
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
authors.forename: Michio
authors.initials: M
authors.lastname: Takashima
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Tsutsumi
authors.affiliation: Chemical Research Laboratories, Kyowa Hakko Kirin Co., Ltd., 1188, Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411-8731, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Okawa
mesh_terms: D002846:Chromatography, Affinity; D003978:Diazomethane / Q000737:chemistry; D008024:Ligands; D008853:Microscopy; D004364:Pharmaceutical Preparations / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D012235:Rhodamines / Q000737:chemistry; D016559:Tacrolimus / Q000737:chemistry / Q000302:isolation & purification; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D008024:Ligands; D004364:Pharmaceutical Preparations; D012235:Rhodamines; D003978:Diazomethane; D022061:Tacrolimus Binding Protein 1A; C029773:rhodamine B; D016559:Tacrolimus
keywords: Affinity purification; Drug target identification; Immobilization; Photochemistry