ABSTRACT
Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.
Subject(s)
Alkanesulfonic Acids/metabolism , Carrier Proteins/metabolism , Fluorocarbons/metabolism , Peptide Library , Alkanesulfonic Acids/chemistry , Amino Acid Sequence , Animals , Cell Line , Computational Biology , Fluorocarbons/chemistry , Humans , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Mice , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Software , Surface Plasmon Resonance , Tumor Necrosis Factor-alpha/metabolismABSTRACT
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.
Subject(s)
Acrylic Resins/chemistry , Alkanes/chemistry , Alkanes/metabolism , Piperidines/chemistry , Piperidines/metabolism , Polyethylene Glycols/chemistry , Tacrolimus Binding Protein 1A/analysis , Tacrolimus Binding Protein 1A/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 , Protein Binding , Substrate Specificity , Tacrolimus Binding Protein 1A/isolation & purificationABSTRACT
Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.