A fluorescence resonance energy transfer-based binding assay for characterizing kinase inhibitors: Important role for C-terminal biotin tagging of the kinase
| Autor: | Joyce Kwan, J. Michael Bradshaw, David E. Shaw, Alden Er Hong Ling, Eva Papp |
|---|---|
| Rok vydání: | 2009 |
| Předmět: |
Streptavidin
Biophysics Biotin Biology Biochemistry Antibodies chemistry.chemical_compound Agammaglobulinaemia Tyrosine Kinase Fluorescence Resonance Energy Transfer Histidine Protein kinase A Protein Kinase Inhibitors Molecular Biology Glutathione Transferase Kinase Ligand binding assay Cell Biology Protein-Tyrosine Kinases Protein Structure Tertiary Förster resonance energy transfer chemistry Biotinylation Oligopeptides Tyrosine kinase |
| Zdroj: | Analytical Biochemistry. 395:256-262 |
| ISSN: | 0003-2697 |
| DOI: | 10.1016/j.ab.2009.08.032 |
| Popis: | Novel biochemical strategies are needed to identify the next generation of protein kinase inhibitors. One promising new assay format is a competition binding approach that employs time-resolved fluorescence resonance energy transfer (TR–FRET). In this assay, a FRET donor is bound to the kinase via a purification tag, whereas a FRET acceptor is bound via a tracer-labeled inhibitor. Displacement of the tracer by an unlabeled inhibitor eliminates FRET between the fluorophores and provides a readout on binding. Although promising, this technique has so far been limited in applicability in part by a lack of signal strength is some cases and also by an inability to predict whether a particular tagging strategy will show robust FRET. In this work, we sought to better understand the factors that give rise to a strong FRET signal in this assay. We determined the magnitude of FRET for several tyrosine kinases using different purification tags (biotin, glutathione S -transferase [GST], and His) placed at either the N terminus or C terminus of the kinase. It was observed that coupling the FRET acceptor to the kinase C terminus using a biotin/streptavidin interaction resulted in the greatest increase in FRET. Specifically, for multiple kinases, the signal/background ratio was at least 3-fold better using C-terminal biotinylation compared with tagging at the N terminus using a His/anti-His antibody or GST/anti-GST antibody interaction. In one case, the FRET signal using C-terminal biotin tagging was more than 150-fold over background. This strong FRET signal facilitated development of improved inhibitor binding assays that required only tens of picomolar enzyme or tracer-labeled inhibitor. Together, these results indicate that C-terminal biotinylation is a promising tagging strategy for developing an optimal FRET-based competition binding assay for tyrosine kinases. |
| Databáze: | OpenAIRE |
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