Expression and purification of soluble single-chain Fv against human fibroblast growth factor receptor 3 fused with Sumo tag in Escherichia coli
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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DOI：
10.1016/j.ejbt.2015.05.006
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发表时间：
2015-07-01
影响因子：
2.7
通讯作者：
Xiao, Yechen
中科院分区：
文献类型：
--
作者：
Liu, Zixuan;Zhang, Jizhou;Xiao, Yechen
背景：成纤维细胞生长因子受体3（Fibroblast growth factor receptor 3，FGFR 3）的过表达或突变激活参与了多种肿瘤的发病机制。抗FGFR 3单链抗体的研究越来越受到重视。结果：成功制备了一种新型抗FGFR 3单链抗体（ScFv）。为获得可溶性表达，采用PCR方法将单链抗体与Sumo（Small ubiquitin-related modifiers）融合，并克隆入pET-20 b中。重组菌经0.5mM异丙基-β-D-硫代半乳糖苷（IPTG）诱导16 h后，收获Sumo-ScFv上清，Ni-NTA层析纯化。经Sumo蛋白酶酶切后，将重组单链抗体从融合蛋白中释放出来，并通过Ni-NTA层析进一步纯化。单链抗体的纯度大于95%，每升细菌培养物中单链抗体的产量可达4 mg。体外实验结果表明，ScFv能显著抑制FGF 9诱导的FGFR 3磷酸化。结论：本研究为在大肠杆菌中获得可溶性表达的ScFv及其生物活性提供了一种新的方法。(C)2015年瓦尔帕莱索天主教大学Elsevier B. V.制作和主持。保留所有权利。
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-beta-D-thiogalactopyranoside (IPTG) for 16 h at 20 degrees 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. (C) 2015 Pontificia Universidad Catolica de Valparaiso. Production and hosting by Elsevier B.V. All rights reserved.