Analysis of Interactions of Signaling Proteins with Phage-Displayed Ligands by Fluorescence Correlation Spectroscopy
| Autor: | Helmi R. M. Schlaman, Herman P. Spaink, Maarten H. van Es, Tjerk H. Oosterkamp, Dorien Ottenhof, Kristiane A. Schmidt |
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| Jazyk: | angličtina |
| Rok vydání: | 2008 |
| Předmět: |
Phage display
Recombinant Fusion Proteins Molecular Sequence Data Peptide Fluorescence correlation spectroscopy Biology Ligands Microscopy Atomic Force Biochemistry Analytical Chemistry Peptide Library Amino Acid Sequence 14-3-3 protein chemistry.chemical_classification Ligand (biochemistry) Fluorescence Crystallography Spectrometry Fluorescence chemistry 14-3-3 Proteins Biophysics Molecular Medicine Titration Peptides Two-dimensional nuclear magnetic resonance spectroscopy Biotechnology Protein Binding Signal Transduction |
| Zdroj: | Slas Discovery, 13(8), 766-776 Slas Discovery |
| Popis: | Fluorescent correlation spectroscopy (FCS) was used to measure binding affinities of ligands to ligates that are expressed by phage-display technology. Using this method we have quantified the binding of the 14-3-3 signaling protein to artificial peptide ligand. As a ligand we used the R18 artificial peptide expressed as a fusion in the cpIII coat protein that is present in 3 to 5 copies in an M13 phage. Comparisons of binding affinities were made with free R18 ligands using FCS. The result showed a relatively high binding affinity for the phage-displayed R18 peptide compared with binding to free fluorescently labeled R18. Quantification was supported by titration of the phage numbers using atomic force microscopy (AFM). AFM was shown to accurately determine phage numbers in solution as a good alternative for electron microscopy. It was shown to give reliable data that correlated perfectly with those of the viable phage numbers determined by classical bacterial infection studies. In conclusion, a very fast and sensitive method for the selection of new peptide ligands or ligates based on a quantitative assay in solution has been developed. (Journal of Biomolecular Screening XXXX:xx-xx) |
| Databáze: | OpenAIRE |
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