Data/hora: 01/06/2024 07:27:04

Provedor de dados: | 69
País: | Chile
Título: | Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
Autores: | Liu,Zixuan; Zhang,Jizhou; Fan,Hongqiong; Yin,Ruofeng; Zheng,Zhong; Xu,Qian; Liu,Qing; He,Haiting; Peng,Xiaofan; Wang,XinXin; Li,Xiaokun; Xiao,Yechen.
Data: | 2015-07-01
Ano: | 2015
Palavras-chave: | Escherichia coli; Fibroblast growth factor receptor 3; Single-chain Fv antibody; Soluble expression; Sumo tag.
Resumo: | Background Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. More and more studies focus on the potential usage of therapeutic antibodies against FGFR3. Results In this study, a novel single-chain Fv (ScFv) against FGFR3 was prepared and characterized. To achieve the soluble expression, ScFv was fused with Sumo (Small ubiquitin-related modifier) by polymerase chain reaction (PCR), and cloned into pET-20b. The recombinant bacteria were induced by 0.5 mM Isopropyl-ß-d-thiogalactopyranoside (IPTG) for 16 h at 20°C, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After being cleaved by the Sumo protease, the recombinant ScFv was released from the fusion protein, and further purified by Ni-NTA chromatography. The purity of ScFv was shown to be higher than 95% and their yield reached 4 mg per liter of bacterial culture. In vitro data showed that ScFv can significantly attenuate FGF9-induced phosphorylation of FGFR3. Conclusion We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli.
Tipo: | Journal article
Idioma: | Inglês
Identificador: | http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000400008
Editor: | Pontificia Universidad Católica de Valparaíso
Formato: | text/html
Fonte: | Electronic Journal of Biotechnology  v.18 n.4 2015