Background: Anti-TNF-α scFv is gaining acceptance as an effective drug for various diseases, such as rheumatoid arthritis and Crohn’s disease that involve elevated levels of TNF-α. The single-chain variable fragment (scFv) consists of variable regions of heavy and light chains of monoclonal antibodies (mAb). Due to its smaller size, it curbs the mAb’s auto-antibody effects and their limitation of penetration into the tissues during the neutralization of TNF-α.

Objective: In this work, a cDNA coding for anti-TNF-α scFv was successfully cloned into a pRSET-B vector and efficiently expressed in an E. coli strain GJ1158, a salt inducible system that uses sodium chloride instead of IPTG as an inducer.

Methods: The protein was expressed in the form of inclusion bodies (IB), solubilized using urea, and refolded by pulse dilution. Further, the amino acid sequence coverage of scFv was confirmed by ESI-Q-TOF MS/MS and MALDI-TOF. Further studies on scaling up the production of scFv and its application of scFv are being carried out.

Results: The soluble fraction of anti-TNF-α scFv was then purified in a single chromatographic step using CM-Sephadex chromatography, a weak cation exchanger with a yield of 10.3 mg/L. The molecular weight of the scFv was found to be ~ 28 kDa by SDS PAGE, and its presence was confirmed by western blot analysis and mass spectrometry.

Conclusion: Anti-TNF-α scFv has been successfully purified in a salt inducible system GJ1158. As per the best of our knowledge, this is the first report of purification of Anti-TNF-α scFv in a salt inducible system from soluble fractions as well as inclusion bodies.

背景：抗 TNF-α scFv 作为治疗各种疾病的有效药物正在获得认可，例如类风湿性关节炎和克罗恩病，这些疾病涉及 TNF-α 水平升高。单链可变片段 (scFv) 由单克隆抗体 (mAb) 的重链和轻链可变区组成。由于其较小的尺寸，它抑制了 mAb 的自身抗体效应及其在中和 TNF-α 期间对组织的渗透限制。

目的：在这项工作中，将编码抗 TNF-α scFv 的 cDNA 成功克隆到 pRSET-B 载体中，并在大肠杆菌菌株 GJ1158 中高效表达，这是一种盐诱导系统，使用氯化钠代替 IPTG 作为诱导剂.

方法：蛋白质以包涵体（IB）的形式表达，用尿素溶解，并通过脉冲稀释重新折叠。此外，通过 ESI-Q-TOF MS/MS 和 MALDI-TOF 证实了 scFv 的氨基酸序列覆盖率。正在进一步研究扩大scFv的生产及其在scFv中的应用。

结果：抗 TNF-α scFv 的可溶部分随后使用 CM-Sephadex 色谱法（一种产率为 10.3 mg/L 的弱阳离子交换剂）在一个色谱步骤中进行纯化。通过 SDS PAGE 发现 scFv 的分子量约为 28 kDa，并且通过蛋白质印迹分析和质谱法证实了它的存在。

结论：抗 TNF-α scFv 已在盐诱导系统 GJ1158 中成功纯化。据我们所知，这是在盐诱导系统中从可溶性部分和包涵体中纯化抗 TNF-α scFv 的第一份报告。