Abstract
Normal 0 21 false false false ES-CL X-NONE X-NONE 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-β-D- thiogalactopyranoside (IPTG) for 16 h at 20oC, and the supernatant liquid of Sumo-ScFv was harvested and purified by Ni-NTA chromatography. After 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 ScFv can significantly attenuate FGF9-induced the phosphorylation of FGFR3. Conclusion: We provide a novel method to produce soluble expression and bioactive functions of ScFv in Escherichia coli . /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:10.0pt; mso-para-margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-ansi-language:ES-CL; mso-fareast-language:EN-US;}
Highlights
Fibroblast growth factors (FGFs), a family of more than 20 known members, play important roles in regulating cell proliferation, differentiation and survival during embryo development and adult stage [1]
Our results showed that Sumo is very helpful for promoting the soluble expression of single-chain Fv (ScFv) in E. coli, and the resulting recombinant bioactive ScFv can be used for therapeutic applications and clinical diagnosis of patients in the future
The Sumo-ScFv protein was diluted to a concentration of 1 mg ml-1. 10U Sumo protease were added to the dilution in salt buffer (20 mM Tris–HCl, pH 7.0), and the mixture was incubated overnight at 4°C, the cleaved sample was applied to a Ni-NTA resin column
Summary
Overexpression or mutated activation of Fibroblast growth factor receptor 3 (FGFR3) is involved in the pathogenesis of many tumors. 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-β-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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