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....

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Published inBioconjugate chemistry Vol. 19; no. 12; pp. 2417 - 2426
Main Authors Kuramochi, Kouji, Miyano, Yuka, Enomoto, Yoshihiro, Takeuchi, Ryo, Ishi, Kazutomo, Takakusagi, Yoichi, Saitoh, Takeki, Fukudome, Keishi, Manita, Daisuke, Takeda, Yoshifumi, Kobayashi, Susumu, Sakaguchi, Kengo, Sugawara, Fumio
Format Journal Article
LanguageEnglish
Published United States American Chemical Society 17.12.2008
Subjects
Acrylic Resins - chemistry
Alkanes - chemistry
Alkanes - metabolism
Amino Acid Sequence
Bacteriophage T7 - genetics
Bacteriophage T7 - metabolism
Binding sites
Cells
Chemical synthesis
Cloning, Molecular
Gene Library
Humans
Jurkat Cells
Kinetics
Ligands
Molecular Sequence Data
Molecules
Peptides
Peptides - analysis
Peptides - chemistry
Peptides - metabolism
Piperidines - chemistry
Piperidines - metabolism
Polyethylene Glycols - chemistry
Protein Binding
Proteins
Resins
Substrate Specificity
Tacrolimus Binding Protein 1A - analysis
Tacrolimus Binding Protein 1A - isolation & purification
Tacrolimus Binding Protein 1A - metabolism
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Summary: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.
Bibliography:istex:E8205E0871AE2DF8ED684B4F258B4EA8C65B8E74
Binding competition assays of FKBP12 absorption on the SLF-immobilized PEGA resin (Figure S1), preparation of lithocolic acid-immobilized resins (Scheme S1), binding analysis between a mammalian DNA polymerase β and the lithocolic acid-immobilized resins by the Bradford method (Figure S2), preparation of glutathione (GSH)-immobilized resins (Scheme S2), binding analysis between glutathione S-transferase (GST) and the glutathione (GSH)-immobilized resins by the Bradford method (Figure S3), and alignment between the clone 2 peptide and human Sox 8−10 proteins (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.
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ISSN:1043-1802
1520-4812
DOI:10.1021/bc8002716