Identification of small molecule binding molecules by affinity purification using a specific ligand immobilized on PEGA resin.
We investigated the application of resins used in solid-phase synthesis for affinity purification. A synthetic ligand for FK506-binding protein 12 (SLF) was immobilized on various resins, and the binding assays between the SLF-immobilized resins and FK506-binding protein 12 (FKBP12) were performed. Of the resins tested in this study, PEGA resin was the most effective for isolating FKBP12. This matrix enabled the isolation of FKBP12 from a cell lysate, and the identification of SLF-binding peptides from a phage cDNA library. We confirmed the interaction between SLF and these peptides using a cuvette type quartz crystal microbalance (QCM) apparatus. Our study suggests that PEGA resin has great potential as a tool not only for the purification and identification of small-molecule binding proteins but also for the selection of peptides that recognize target molecules.
Kuramochi
Kouji
K
Department of Applied Biological Science, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.
Miyano
Yuka
Y
Enomoto
Yoshihiro
Y
Takeuchi
Ryo
R
Ishi
Kazutomo
K
Takakusagi
Yoichi
Y
Saitoh
Takeki
T
Fukudome
Keishi
K
Manita
Daisuke
D
Takeda
Yoshifumi
Y
Kobayashi
Susumu
S
Sakaguchi
Kengo
K
Sugawara
Fumio
F
D000180:Acrylic Resins / Q000737:chemistry*; D000473:Alkanes / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D017123:Bacteriophage T7 / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D019169:Jurkat Cells; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010880:Piperidines / Q000737:chemistry* / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D013379:Substrate Specificity; D022061:Tacrolimus Binding Protein 1A / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism*
C511576:AP1497; D000180:Acrylic Resins; D000473:Alkanes; D008024:Ligands; D010455:Peptides; D010880:Piperidines; C520358:poly(acryloyl-bis(aminopropyl)polyethylene glycol); D011092:Polyethylene Glycols; D022061:Tacrolimus Binding Protein 1A