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Electron. j. biotechnol ; 18(4): 302-306, July 2015. ilus
Artigo em Inglês | LILACS | ID: lil-757868

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.


Assuntos
Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Anticorpos de Cadeia Única/isolamento & purificação , Anticorpos de Cadeia Única/metabolismo , Solubilidade , Espectrometria de Massas , Proteínas Recombinantes , Western Blotting , Escherichia coli
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