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Protein & Peptide Letters

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ISSN (Print): 0929-8665
ISSN (Online): 1875-5305

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Research Article

Study on Cloning and Expression of TNF-α Variants in E. coli: Production, Purification, and Interaction with Anti-TNF-α Inhibitors

Author(s): Gülşah Akçadağ, Demet Cansaran-Duman*, Emine Sümer Aras* and Haluk Ataoğlu

Volume 31, Issue 5, 2024

Published on: 05 June, 2024

Page: [395 - 408] Pages: 14

DOI: 10.2174/0109298665312592240516111404

Abstract

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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