在大肠杆菌中克隆和表达 TNF-α 变体的研究:生产、纯化及与抗肿瘤坏死因子-α抑制剂的相互作用。

IF 1 4区 生物学 Q4 BIOCHEMISTRY & MOLECULAR BIOLOGY Protein and Peptide Letters Pub Date : 2024-01-01 DOI:10.2174/0109298665312592240516111404
Gülşah Akçadağ, Demet Cansaran-Duman, Emine Sümer Aras, Haluk Ataoğlu
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引用次数: 0

摘要

背景: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。大剂量给药时,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,它能与各种抗 TNF-α 抑制剂高效相互作用:方法:利用重组 DNA 技术将三种不同版本的密码子优化人 TNF-α 序列克隆到大肠杆菌中。采用菌落 PCR 方案进行验证,并通过 SDS-PAGE 和 Western 印迹对产生的蛋白质进行分析。使用尺寸排阻色谱法纯化 sTNF-α。使用酶联免疫吸附技术分析和比较 sTNF-α 与三种不同标准品的结合效率:结果:在原生条件(25°C)下,与阳性对照相比,sTNF-α 与抗 TNF-α 抗体之间的相互作用为 3 970。在变性条件(37°C)下,TNF-α 和抗 TNF-α 抗体之间的相互作用为 0,587 ,而在变性条件(37°C)下,TNF-α 和抗 TNF-α 抗体之间的相互作用为 0,535 。与商用 TNF-α 相比,sTNF-α(920 μg/mL)的 F7 与阿达木单抗、依那西普和英夫利昔单抗的结合效率相同或更高: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。这项研究证明,sTNF-α 与阿达木单抗、依那西普和英夫利昔单抗的结合效率很高,这支持了将其用于治疗的可行性,有助于建立更可持续、更具成本效益和独立的医疗保健系统。
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Study on Cloning and Expression of TNF-α Variants in E. coli: Production, Purification, and Interaction with Anti-TNF-α Inhibitors.

Background: 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.

Objectives: 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.

Methods: Recombinant DNA technology was used to clone three different versions of codon-optimized human TNF-α sequences to E. coli. 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.

Results: 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.

Conclusion: 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.

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来源期刊
Protein and Peptide Letters
Protein and Peptide Letters 生物-生化与分子生物学
CiteScore
2.90
自引率
0.00%
发文量
98
审稿时长
2 months
期刊介绍: Protein & Peptide Letters publishes letters, original research papers, mini-reviews and guest edited issues in all important aspects of protein and peptide research, including structural studies, advances in recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, and drug design. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallization and preliminary structure determination of biologically important proteins are considered only if they include significant new approaches or deal with proteins of immediate importance, and preliminary structure determinations of biologically important proteins. Purely theoretical/review papers should provide new insight into the principles of protein/peptide structure and function. Manuscripts describing computational work should include some experimental data to provide confirmation of the results of calculations. Protein & Peptide Letters focuses on: Structure Studies Advances in Recombinant Expression Drug Design Chemical Synthesis Function Pharmacology Enzymology Conformational Analysis Immunology Biotechnology Protein Engineering Protein Folding Sequencing Molecular Recognition Purification and Analysis
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