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Analysis of Interactions of Signaling Proteins with Phage-Displayed Ligands by Fluorescence Correlation Spectroscopy

Institute of Biology Leiden, Leiden University, Clusius Laboratory, Leiden, The Netherlands

Kristiane Schmidt

Institute of Biology Leiden, Leiden University, Clusius Laboratory, Leiden, The Netherlands, Philips Research, Care & Health Applications, High Tech Campus 12a, Room WAD p.101 (box WAD 01), 5656 AE Eindhoven, The Netherlands

Dorien Ottenhof

Leiden Institute of Physics, Leiden University, Huygens Laboratory, Leiden, The Netherlands

Maarten H. van Es

Leiden Institute of Physics, Leiden University, Huygens Laboratory, Leiden, The Netherlands

Tjerk H. Oosterkamp

Leiden Institute of Physics, Leiden University, Huygens Laboratory, Leiden, The Netherlands

Herman P. Spaink

Institute of Biology Leiden, Leiden University, Clusius Laboratory, Leiden, The Netherlands, h.p.spaink{at}biology.leidenuniv.nl

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 2008:766-776)

Key Words: phage display • fluorescence correlation spectroscopy • atomic force microscopy • 14-3-3 protein • binding affinity

This version was published on September 1, 2008

Journal of Biomolecular Screening, Vol. 13, No. 8, 766-776 (2008)
DOI: 10.1177/1087057108323124


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