Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner
Soluble expression of single-chain variable fragment (scFv) in Escherichia coli using superfolder green fluorescent protein as fusion partner
复制标题
使用超级文件夹绿色荧光蛋白作为融合伴侣在大肠杆菌中可溶性表达单链可变片段（scFv）
DOI：
10.1007/s00253-019-09925-6
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发表时间：
2019-08-01
影响因子：
5
通讯作者：
Yu, Xiaolan
中科院分区：
文献类型：
--
作者：
Liu, Min;Wang, Bin;Yu, Xiaolan
单链抗体（Single-chain variable fragment，scFv）由于其结合亲和性和低免疫原性，在医学治疗和诊断应用中具有广阔的前景。然而，scFv的应用受到其异源表达面临的不溶性聚集的挑战的限制。sfGFP已被开发为融合标签，以促进融合伴侣在大肠杆菌中的溶解性。我们在sfGFP的C端设计了抗流感病毒PB 2单链抗体的融合蛋白，并成功地在大肠杆菌中获得了可溶性表达。在18 °C下用0.1mM IPTG诱导16 h，sfGFP-scFv-His的表达量达到20 mg/L。用TEV蛋白酶切割10 mg纯sfGFP-scFv-His，得到6 mg scFv-His。另外，我们发现sfGFP-scFv-His在鸡血清中比scFv-His更稳定，提示sfGFP不仅有利于scFv在大肠杆菌中的溶解，而且促进了scFv的稳定性。Western blot和ELISA检测结果表明，sfGFP-scFv-His具有特异性结合PB 2的免疫活性。血凝试验和实时荧光定量PCR检测结果表明，sfGFP-scFv-His和scFv-His均能抑制H1N1流感病毒在A549细胞中的复制。这些结果进一步发展了单链抗体作为抗流感病毒药物的应用。此外，使用sfGFP作为融合伴侣的scFv的可溶性表达提供了用于制造抗大流行性疾病的scFv的成本有效的制备模型。
Single-chain variable fragment (scFv) has great prospect in medical therapies and diagnostic applications due to its binding affinity and low immunogenicity. However, the application of scFv is limited by its heterologous expression facing challenges of insoluble aggregation. sfGFP has been developed as fusion tag to facilitate the solubility of fusion partner in Escherichia coli. We designed fusion protein of anti-influenza PB2 scFv at C-terminus of sfGFP and successfully obtained soluble expression of sfGFP-scFv-His in Escherichia coli. The expression level of sfGFP-scFv-His reached at 20 mg/L of bacterial culture when the culture was induced with 0.1 mM IPTG at 18 °C for 16 h. And 6 mg scFv-His was obtained from the cleavage of 10 mg pure sfGFP-scFv-His with TEV protease. In addition, we found that sfGFP-scFv-His was more stable than scFv-His in chicken serum, suggesting that sfGFP not only facilitated the solubility of scFv in Escherichia coli, but also promoted the stability of scFv. The immunologic activity of sfGFP-scFv-His was confirmed by Western blot and ELISA; the results showed that anti-PB2 sfGFP-scFv-His exhibited specific binding to PB2. Hemagglutination and comparative real-time RT-PCR analysis indicated that sfGFP-scFv-His and scFv-His inhibited the replication of H1N1 influenza virus in the infected A549 cells. These results further develop the application of scFv as an agent, such as anti-influenza. Furthermore, soluble expression of scFv using sfGFP as fusion partner provide a cost-effective preparation model for manufacturing scFv against pandemic disease.