title: High-level expression, purification, and characterization of bifunctional ScFv-9R fusion protein.
abstract: Fibroblast growth factor receptor 3 (FGFR3) is a noted proto-oncogene involved in the pathogenesis of many tumors, so more and more studies focus on the potential use of receptor kinase inhibitor and therapeutic antibodies against FGFR3. In this study, we designed a novel fusion protein containing the single-chain Fv (ScFv) against FGFR3 and 9-arginine, denoted as ScFv-9R. To achieve the high-level production and soluble expression, ScFv and ScFv-9R were fused with small ubiquitin-related modifier (Sumo) by polymerase chain reaction and expressed in Escherichia coli BL21 (DE3). The recombinant bacteria was induced by 0.5 mM isopropyl-β-D-thiogalactopyranoside for 20 h at 20 °C; supernatants of Sumo-ScFv was harvested and purified by DEAE Sepharose FF and Ni-NTA orderly, and supernatants of Sumo-ScFv-9R was harvested and purified by Ni-NTA. After cleaved by the Sumo protease, the recombinant ScFv or ScFv-9R was released from the fusion protein, respectively. The purity of ScFv or ScFV-9R was shown to be higher than 90 %, and their yield reached 3-5 mg per liter of bacterial culture. In vitro data showed that ScFV-9R can attenuate the phosphorylation of FGFR3 and ERK in the absence or presence of FGF9. Gel retardation assay showed that 1 μg of ScFv-9R could efficiently bind to about 4 pmol siRNA. Fluorescent microscope analysis showed that ScFv-9R can efficiently bind and deliver siRNA into RT112 cells. In conclusion, we use Sumo fusion system to acquire high-level production, soluble expression, and bifunctional activity of ScFv-9R in E. coli. Our results also revealed that ScFv-9R, as a novel carrier, may have potential applications in antitumor studies and pharmaceutical development.
authors: Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, Changchun, 130021, China.
authors: Xiguang
authors: X
authors: Zhang
authors: Jiasen
authors: J
authors: Xie
authors: Yan
authors: Y
authors: Sun
authors: Huijing
authors: H
authors: Xu
authors: Tonghua
authors: T
authors: Du
authors: Zixuan
authors: Z
authors: Liu
authors: Jinhui
authors: J
authors: Chen
authors: Zhong
authors: Z
authors: Zheng
authors: Keqiang
authors: K
authors: Liu
authors: Jizhou
authors: J
authors: Zhang
authors: Mujie
authors: M
authors: Kan
authors: Xiaokun
authors: X
authors: Li
authors: Yechen
authors: Y
authors: Xiao
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D000090063:Proto-Oncogene Mas; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D025841:Small Ubiquitin-Related Modifier Proteins; D051498:Receptor, Fibroblast Growth Factor, Type 3