Development of an ELISA-based high throughput screening method for novel anticancer agents targeting β-catenin/Lef1 interaction / 生物工程学报
Chinese Journal of Biotechnology
;
(12): 707-717, 2019.
Artículo
en Chino
| WPRIM
| ID: wpr-771339
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.
Texto completo:
Disponible
Índice:
WPRIM (Pacífico Occidental)
Asunto principal:
Ensayo de Inmunoadsorción Enzimática
/
Beta Catenina
/
Factor de Unión 1 al Potenciador Linfoide
/
Ensayos Analíticos de Alto Rendimiento
/
Antineoplásicos
Tipo de estudio:
Estudio diagnóstico
/
Estudio pronóstico
/
Estudio de tamizaje
Idioma:
Chino
Revista:
Chinese Journal of Biotechnology
Año:
2019
Tipo del documento:
Artículo
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