Department of Biology, Science Faculty, Ankara University, Tandogan, Ankara, Turkey.
Matriks Biotechnology Industry and Trade Ltd. Co., Gazi Teknopark, Ankara, Turkey.
Protein Pept Lett. 2024;31(5):395-408. doi: 10.2174/0109298665312592240516111404.
TNF-α is a proinflammatory cytokine and plays a role in cell proliferation, differentiation, survival, and death pathways. When administered at high doses, it may cause damage to the tumor vasculature, thereby increasing the permeability of the blood vessels. Therefore, monitoring the dose and the response of the TNF-α molecule is essential for patients' health.
This study aimed to clone, express, and purify the active form of the TNF-α protein, which can interact with various anti-TNF-α inhibitors with high efficiency.
Recombinant DNA technology was used to clone three different versions of codon-optimized human TNF-α sequences to Colony PCR protocol was used for verification and produced proteins were analyzed through SDS-PAGE and western blot. Size exclusion chromatography was used to purify sTNF-α. ELISA techniques were used to analyze and compare binding efficiency of sTNF-α against three different standards.
Under native condition (25°C), interaction between sTNF-α and anti-TNF-α antibody was 3,970, compared to positive control. The interaction was 0,587, whereas it was 0,535 for TNF- α and anti-TNF-α antibodies under denaturing conditions (37°C). F7 of sTNF-α (920 μg/mL) had the same/higher binding efficiency to adalimumab, etanercept, and infliximab, compared to commercial TNF-α.
This study was the first to analyze binding efficiency of homemade sTNF-α protein against three major TNF-α inhibitors (adalimumab, etanercept, and infliximab) in a single study. The high binding efficiency of sTNF-α with adalimumab, etanercept, and infliximab, evidenced in this study supports the feasibility of its use in therapeutic applications, contributing to more sustainable, cost-effective, and independent healthcare system.
TNF-α 是一种促炎细胞因子，在细胞增殖、分化、存活和死亡途径中发挥作用。当以高剂量给药时，它可能会对肿瘤血管造成损伤，从而增加血管的通透性。因此，监测 TNF-α 分子的剂量和反应对患者的健康至关重要。
本研究旨在克隆、表达和纯化 TNF-α 蛋白的活性形式，该蛋白可以与各种高效的抗 TNF-α 抑制剂相互作用。
使用重组 DNA 技术克隆了三种不同版本的密码子优化的人 TNF-α 序列。使用集落 PCR 方案进行验证，并通过 SDS-PAGE 和 Western blot 分析产生的蛋白质。使用尺寸排阻层析法纯化 sTNF-α。使用 ELISA 技术分析和比较 sTNF-α 与三种不同标准品的结合效率。
在天然条件（25°C）下，sTNF-α与抗 TNF-α 抗体的相互作用为 3.970，与阳性对照相比。相互作用为 0.587，而在变性条件（37°C）下，TNF-α与抗 TNF-α 抗体的相互作用为 0.535。与商业 TNF-α相比，sTNF-α 的 F7（920μg/mL）对阿达木单抗、依那西普和英夫利昔单抗具有相同/更高的结合效率。
本研究首次在一项研究中分析了自制 sTNF-α 蛋白与三种主要 TNF-α 抑制剂（阿达木单抗、依那西普和英夫利昔单抗）的结合效率。本研究中 sTNF-α 与阿达木单抗、依那西普和英夫利昔单抗的高结合效率支持其在治疗应用中的可行性，有助于建立更可持续、更具成本效益和更独立的医疗保健系统。
