Selective ligand purification using high-performance affinity beads.
Since the development of affinity chromatography, affinity purification technology has been applied to many aspects of biological research, becoming an indispensable tool. Efficient strategies for the identification of biologically active compounds based on biochemical specificity have not yet been established, despite widespread interest in identifying chemicals that directly alter biomolecular functions. Here, we report a novel method for purifying chemicals that specifically interact with a target biomolecule using reverse affinity beads, a receptor-immobilized high-performance solid-phase matrix. When FK506-binding protein 12 (FKBP12) immobilized beads were used in this process, FK506 was efficiently purified in one step either from a mixture of chemical compounds or from fermented broth extract. The reverse affinity beads facilitated identification of drug/receptor complex binding proteins by reconstitution of immobilized ligand/receptor complexes on the beads. When FKBP12/FK506 and FKBP12/rapamycin complexes were immobilized, calcineurin and FKBP/rapamycin-associated protein were purified from a crude cell extract, respectively. These data indicate that reverse affinity beads are powerful tools for identification of both specific ligands and proteins that interact with receptor/ligand complexes.
Ohtsu
Yoshihiro
Y
Frontier Collaborative Research Center, Tokyo Institute of Technology, Yokohama 226-8503, Japan.
Ohba
Reiko
R
Imamura
Yoshimasa
Y
Kobayashi
Motoo
M
Hatori
Hidetaka
H
Zenkoh
Tatsuya
T
Hatakeyama
Mamoru
M
Manabe
Takashi
T
Hino
Motohiro
M
Yamaguchi
Yuki
Y
Kataoka
Kohsuke
K
Kawaguchi
Haruma
H
Watanabe
Hajime
H
Handa
Hiroshi
H
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008863:Microspheres; D051381:Rats; D016559:Tacrolimus / Q000737:chemistry / Q000302:isolation & purification*; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry*
D008024:Ligands; D022061:Tacrolimus Binding Protein 1A; D016559:Tacrolimus