Zhang Xiguang, Xie Jiasen, Sun Yan, Xu Huijing, Du Tonghua, Liu Zixuan, Chen Jinhui, Zheng Zhong, Liu Keqiang, Zhang Jizhou, Kan Mujie, Li Xiaokun, Xiao Yechen
Department of Biochemistry and Molecular Biology, College of Basic Medical Science, Jilin University, Changchun, 130021, China.
Appl Microbiol Biotechnol. 2014 Jun;98(12):5499-506. doi: 10.1007/s00253-014-5541-x. Epub 2014 Feb 12.
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.
成纤维细胞生长因子受体3（FGFR3）是一种著名的原癌基因，参与多种肿瘤的发病机制，因此越来越多的研究聚焦于针对FGFR3的受体激酶抑制剂和治疗性抗体的潜在用途。在本研究中，我们设计了一种新型融合蛋白，其包含针对FGFR3的单链Fv（ScFv）和9-精氨酸，命名为ScFv-9R。为实现高水平生产和可溶性表达，通过聚合酶链反应将ScFv和ScFv-9R与小泛素相关修饰物（Sumo）融合，并在大肠杆菌BL21（DE3）中表达。重组菌在20℃下用0.5 mM异丙基-β-D-硫代半乳糖苷诱导20小时；依次通过DEAE Sepharose FF和Ni-NTA收获并纯化Sumo-ScFv的上清液，通过Ni-NTA收获并纯化Sumo-ScFv-9R的上清液。用Sumo蛋白酶切割后，重组ScFv或ScFv-9R分别从融合蛋白中释放出来。ScFv或ScFV-9R的纯度高于90%，其产量达到每升细菌培养物3-5毫克。体外数据表明，在存在或不存在FGF9的情况下，ScFV-9R均可减弱FGFR3和ERK的磷酸化。凝胶阻滞试验表明，1μg ScFv-9R可有效结合约4 pmol siRNA。荧光显微镜分析表明，ScFv-9R可有效结合并将siRNA递送至RT112细胞中。总之，我们利用Sumo融合系统在大肠杆菌中实现了ScFv-9R的高水平生产、可溶性表达和双功能活性。我们的结果还表明，ScFv-9R作为一种新型载体，可能在抗肿瘤研究和药物开发中具有潜在应用。
