Akademik Veri Yönetim Sistemi
PROTEIN AND PEPTIDE LETTERS, 2024 (SCI-Expanded)
Background: TNF-alpha 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-alpha molecule is essential for patients' health. Objectives: This study aimed to clone, express, and purify the active form of the TNF-alpha protein, which can interact with various anti-TNF-alpha inhibitors with high efficiency. Methods: Recombinant DNA technology was used to clone three different versions of codon-optimized human TNF-alpha 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-alpha. ELISA techniques were used to analyze and compare binding efficiency of sTNF-alpha against three different standards. Results: Under native condition (25 degrees C), interaction between sTNF-alpha and anti-TNF-alpha antibody was 3,970, compared to positive control. The interaction was 0,587, whereas it was 0,535 for TNF-alpha and anti-TNF-alpha antibodies under denaturing conditions (37 degrees C). F7 of sTNF-alpha (920 mu g/mL) had the same/higher binding efficiency to adalimumab, etanercept, and infliximab, compared to commercial TNF-alpha. Conclusion: This study was the first to analyze binding efficiency of homemade sTNF-alpha protein against three major TNF-alpha inhibitors (adalimumab, etanercept, and infliximab) in a single study. The high binding efficiency of sTNF-alpha 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.