Development of the high-throughput screening fluorescence polarization assay for HSP90 inhibitor
10.3969/j.issn.1005-1678.2017.02.003
- VernacularTitle:采用荧光偏振法建立HSP90抑制剂的高通量筛选模型
- Author:
Nina XUE
;
Chunyang WANG
;
Hanze YANG
;
Yue CHEN
;
Jing JIN
;
Xiaoguang CHEN
- Keywords:
fluorescence polarization;
HSP90 inhibitor;
protein expression and purification
- From:
Chinese Journal of Biochemical Pharmaceutics
2017;37(2):8-11
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish the high-throughput screening fluorescence polarization assay for HSP90 inhibitor.Methods E.coli strain BL21 ( DE3) competent cells were transformed with pET24α( +)-HSP90αplasmid.The cell lysate supernatant was induced to product the soluble protein and purified with Ni-NTA agarose.Western blot analysis was used to identify whether the purified protein is HSP90α.The fluorescence polarization assay for screening HSP90 inhibitors was established and optimized using varying concentrations of recombinant HSP90 protein and molecular probe VER00051001.Meanwhile, the binding activity of GA and NVP-AUY922 for HSP90αwas measured by fluorescence polarization assay.Results HSP90αwas induced expression and purified successfully.The fluorescence polarization assay was performed using 80 nM probe VER00051001 and 2.01μg/mL HSP90α, with the Z factor of 0.83.GA and NVP-AUY922 competed with the probes VER00051001 for binding sites of HSP90, with IC50 of 55 nM and 13 nM, respectively.Conclusion A reliable model was established using fluorescence polarization assay for screening HSP90 inhibitors.
