Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

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 FGF...

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Autores principales:	Liu,Zixuan, Zhang,Jizhou, Fan,Hongqiong, Yin,Ruofeng, Zheng,Zhong, Xu,Qian, Liu,Qing, He,Haiting, Peng,Xiaofan, Wang,XinXin, Li,Xiaokun, Xiao,Yechen
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2015
Materias:	Escherichia coli Fibroblast growth factor receptor 3 Single-chain Fv antibody Soluble expression Sumo tag
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000400008
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spelling	oai:scielo:S0717-345820150004000082015-12-04Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coliLiu,ZixuanZhang,JizhouFan,HongqiongYin,RuofengZheng,ZhongXu,QianLiu,QingHe,HaitingPeng,XiaofanWang,XinXinLi,XiaokunXiao,Yechen Escherichia coli Fibroblast growth factor receptor 3 Single-chain Fv antibody Soluble expression Sumo tag 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.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.18 n.4 20152015-07-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000400008en10.1016/j.ejbt.2015.05.006
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	Escherichia coli Fibroblast growth factor receptor 3 Single-chain Fv antibody Soluble expression Sumo tag
spellingShingle	Escherichia coli Fibroblast growth factor receptor 3 Single-chain Fv antibody Soluble expression Sumo tag Liu,Zixuan Zhang,Jizhou Fan,Hongqiong Yin,Ruofeng Zheng,Zhong Xu,Qian Liu,Qing He,Haiting Peng,Xiaofan Wang,XinXin Li,Xiaokun Xiao,Yechen Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
description	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.
author	Liu,Zixuan Zhang,Jizhou Fan,Hongqiong Yin,Ruofeng Zheng,Zhong Xu,Qian Liu,Qing He,Haiting Peng,Xiaofan Wang,XinXin Li,Xiaokun Xiao,Yechen
author_facet	Liu,Zixuan Zhang,Jizhou Fan,Hongqiong Yin,Ruofeng Zheng,Zhong Xu,Qian Liu,Qing He,Haiting Peng,Xiaofan Wang,XinXin Li,Xiaokun Xiao,Yechen
author_sort	Liu,Zixuan
title	Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
title_short	Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
title_full	Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
title_fullStr	Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
title_full_unstemmed	Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
title_sort	expression and purification of soluble single-chain fv against human fibroblast growth factor receptor 3 fused with sumo tag in escherichia coli
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2015
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582015000400008
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Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli

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