Tubulin degradation agent CRSMs and application thereof
A technology of tubulin and degradation agent, which is applied in the field of protein degradation and achieves good application prospects
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Embodiment 1
[0043] Example 1 Tubulin degradation agent CRSMs binds to the colchicine site to promote tubulin degradation
[0044] The method of competition binding experiment was adopted. The Hela growing in the logarithmic phase was selected and spread on the 96-well plate on which the slide (cover glass) had been laid, and continued to culture for 24 hours until the cells were completely adhered to the wall. Then add 1 μM colchicine (col), pironetin (Pir), vinblastine (Vin) and maytansinoid (May) to incubate the cells for 1 hour, and then add tubulin degrading agent to continue to incubate for 16 hours , total protein was extracted, and western detection of tubulin degradation. The result is as figure 1 As shown, only the α / β-tubulin band at the colchicine site was observed, indicating that the tubulin degradation agent acts on the colchicine site. The bands of α / β-tubulin in cells treated with Pir, Vin and May disappeared, indicating that CRSMs, a tubulin degrading agent, can promot...
Embodiment 2
[0045] Example 2 Structural Biology Experiments to Investigate the Binding Mode of Tubulin Degrading Agents at the Colchicine Site
[0046] Protein expression and purification refer to the reported literature steps, RB3-SLD (RB3 protein stathmin-like domain) was expressed in Escherichia coli, and purified by anion exchange chromatography, then in 10mM HEPES-NaOH (150mMNaCl, 2mM DTT, pH =7.2) through gel filtration. The purified RB3-SLD protein was concentrated to 10 mg / mL and stored in a -80°C refrigerator. His-tagged TTL (recombinant protein) was also expressed in E. coli by Ni 2+ Affinity chromatography column was purified, followed by 20mM Bis propane buffer (200mM NaCl, 2.5mM MgCl 2 , 5 mM β-mercaptoethanol, 1% glycerol, pH = 6.5) with gel filtration. Finally concentrated to 20 mg / mL and stored in -80°C freezer. The purity of RB3 and TTL was checked by SDS-PAGE. Purchased porcine brain tubulin at a concentration of 10 mg / mL was dissolved in GTB buffer and stored at -8...
Embodiment 3
[0047] Example 3 Immunofluorescence experiment to investigate the effect of tubulin degrading agent on microtubule morphology
[0048] Microtubules play an important role in the process of cell mitosis. Microtubule-targeting agents can interfere with cell mitosis, block mitosis at a specific period and induce apoptosis, thereby exerting anti-tumor effects. While microtubule stabilizers such as paclitaxel promote the polymerization of microtubules during mitosis, microtubule destabilizers such as vincristine and colchicine promote the depolymerization of microtubules during mitosis. The morphological changes produced under the interference of targeting agents can be clearly and intuitively observed by immunofluorescence experiments. Therefore, we investigated the effects of tubulin degrading agents on microtubule morphological changes in HeLa cells.
[0049] The HeLa growing in the logarithmic phase was selected and spread on the 6-well plate on which the slide (cover glass) h...
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