Development of an ELISA-based high throughput screening method for novel anticancer agents targeting β-catenin/Lef1 interaction.
- Author:
Yunyu CHEN
1
;
Xiayi NIU
1
;
Yan LI
2
;
Xiaoping LIU
1
Author Information
- Publication Type:Journal Article
- Keywords: ELISA; Wnt inhibitors; high throughput screening; β-catenin; β-catenin/Lef1 interaction
- MeSH: Antineoplastic Agents; Enzyme-Linked Immunosorbent Assay; High-Throughput Screening Assays; Lymphoid Enhancer-Binding Factor 1; beta Catenin
- From: Chinese Journal of Biotechnology 2019;35(4):707-717
- CountryChina
- Language:Chinese
- Abstract: To develop an enzyme-linked immunosorbent assay (ELISA)-based high throughput screening (HTS) method for β-catenin/Lef1 interaction antagonists screening, Escherichia coli Rosetta (DE3) competent cells were transformed with β-catenin-pET-30a(+) plasmid. β-catenin protein was expressed after induction and purified using affinity chromatography. The biological activity of purified β-catenin was further analyzed by GST Pulldown assay. The β-catenin/GST-Lef1 binding model was established using ELISA principle, and the ELISA-based HTS method was further optimized through determination of an optimal coated concentration of GST-Lef1 and working concentration of β-catenin. The results showed that β-catenin protein was successfully expressed and purified. The GST Pulldown assay demonstrated a perfect biological activity for purified β-catenin. Subsequently, the ELISA-based HTS method was performed using 10 μg/mL GST-Lef1 and 6 μg/mL β-catenin, with the Z factor of 0.76. Taken together, we have successfully developed a simple, robust and reliable ELISA-based HTS method for screening of novel Wnt inhibitors targeting β-catenin/Lef1 interaction.
