Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
Electron. j. biotechnol
; 18(4): 302-306, July 2015. ilus
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| LILACS
| ID: lil-757868
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ABSTRACT
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.
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01-internacional
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LILACS
Assunto principal:
Receptor Tipo 3 de Fator de Crescimento de Fibroblastos
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Idioma:
En
Ano de publicação:
2015
Tipo de documento:
Article