Identification of small molecule binding molecules by affinity purification using a specific ligand immobilized on PEGA resin

Bioconjug Chem. 2008 Dec;19(12):2417-26. doi: 10.1021/bc8002716.

Abstract

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.

MeSH terms

  • Acrylic Resins / chemistry*
  • Alkanes / chemistry*
  • Alkanes / metabolism*
  • Amino Acid Sequence
  • Bacteriophage T7 / genetics
  • Bacteriophage T7 / metabolism
  • Cloning, Molecular
  • Gene Library
  • Humans
  • Jurkat Cells
  • Kinetics
  • Ligands
  • Molecular Sequence Data
  • Peptides / analysis
  • Peptides / chemistry
  • Peptides / metabolism
  • Piperidines / chemistry*
  • Piperidines / metabolism*
  • Polyethylene Glycols / chemistry*
  • Protein Binding
  • Substrate Specificity
  • Tacrolimus Binding Protein 1A / analysis*
  • Tacrolimus Binding Protein 1A / isolation & purification
  • Tacrolimus Binding Protein 1A / metabolism*

Substances

  • AP1497
  • Acrylic Resins
  • Alkanes
  • Ligands
  • Peptides
  • Piperidines
  • poly(acryloyl-bis(aminopropyl)polyethylene glycol)
  • Polyethylene Glycols
  • Tacrolimus Binding Protein 1A