A fluorescence correlation spectroscopy-based assay for fragment screening of slowly inhibiting protein–peptide interaction inhibitors
Section snippets
Preparation of c-Cbl TKB domain (24–349)
The DNA fragment encoding the TKB domain of mouse c-Cbl (24–349) (P22682.2) was cloned into the TOPO vector (Life Technologies) as a fusion fragment with an N-terminal native His affinity tag and a TEV protease cleavage site [14]. The protein was synthesized by a cell-free expression system [14]. The obtained crude protein sample was applied to a HisTrap HP column (GE Healthcare) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) containing 100 mM NaCl and 20 mM imidazole on an ÄKTA explorer system
FCS-based binding assay
First, we measured the binding of APSt to c-Cbl by FCS. Final 1 nM APSt and 0–30 μM c-Cbl were mixed, and FCS data of the binding mixtures were obtained. Fig. 1A shows FCS data measured with samples of the free form of the probe (left curve), the c-Cbl-bound form of the probe (right curve), and their mixture (middle curve). Fig. 1B shows the fit of the FCS data. After the fit, percentage ratios of the c-Cbl binding form were plotted (Fig. 2). As shown in Fig. 2, the amounts of the binding form of
Acknowledgments
We thank Noriko Kato and Mitsushiro Yamaguchi (Olympus) and Koji Kuruma (Fujifilm) for their contributions. This work was supported in part by the Target Protein Research Programs of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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