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.
Artigo
em Chinês
| 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:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Assunto principal:
Ensaio de Imunoadsorção Enzimática
/
Beta Catenina
/
Fator 1 de Ligação ao Facilitador Linfoide
/
Ensaios de Triagem em Larga Escala
/
Antineoplásicos
Tipo de estudo:
Estudo diagnóstico
/
Estudo prognóstico
/
Estudo de rastreamento
Idioma:
Chinês
Revista:
Chinese Journal of Biotechnology
Ano de publicação:
2019
Tipo de documento:
Artigo
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