Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3
Section snippets
Peptide synthesis
Peptides were synthesized and purified using standard Fmoc chemistry by the core facility of the Max Planck Institute of Biochemistry (Martinsried, Germany). Coupling to 5-carboxyfluorescein was performed via N,N′-diisopropylcarbodiimide (DIC)/1-hydroxy-benzotriazole (HOBt) activation in N,N-dimethylformamide (DMF) [30]. Unless stated otherwise, peptides were synthesized with an N-terminal amino group and a C-terminal carboxyl group. Peptides were analyzed by high-performance liquid
Results and discussion
To design fluorescent-labeled peptides that can serve as probes in fluorescence polarization assays for the PBDs of Plk2 and Plk3, we turned to the results of peptide library screens for recognition motifs of the PBDs of Plk1, Plk2, and Plk3 [16]. The peptide motif PMQTSpTPK had been reported as a preferred binding motif for the PBD of Plk2 from an immobilized peptide library described by the general sequence MAXXXXSpTXXXXAKK [16]. N-terminal labeling of the preferred binding motif with
Acknowledgments
This work was generously supported by the Department of Molecular Biology (director: Axel Ullrich) at the Max Planck Institute of Biochemistry and by the Bundesministerium für Bildung und Forschung (NGFN-2, Grant 01GS0453 to K.S. and Grant 01GS0451 to T.B.). We extend our thanks to Judith Müller for experimental support.
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