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
Article
in English
| LILACS
| ID: lil-757868
Responsible library:
CL1.1
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.
Full text:
Available
Collection:
International databases
Health context:
Neglected Diseases
Health problem:
Neglected Diseases
/
Zoonoses
Database:
LILACS
Main subject:
Receptor, Fibroblast Growth Factor, Type 3
/
Single-Chain Antibodies
Language:
English
Journal:
Electron. j. biotechnol
Journal subject:
Biotechnology
Year:
2015
Document type:
Article
Affiliation country:
China
Institution/Affiliation country:
Jilin University/CN