Investigation of the production, purification and binding ability of anti-TNF-α scFv J43 antibody identified by phage display technique

Abstract

Introduction: Physiologically TNF- is involved in different immune responses. However, this pro-inflammatory cytokine plays important roles in pathogenesis of various autoimmune diseases like rheumatoid arthritis. Therefore, inhibition of TNF- can be an effective treatment for such pathological states. Aims: In this study, we aimed to investigate the production, purification and binding ability of anti-TNF-α scFv J43 antibody, identified by phage display technique. Material and Methods: The DNA sequence of J43 scFv antibody was modified to change stop codon to tyrosine codon with site directed mutagenesis and then the corrected sequence was cloned into pET28a. The constructed vector was transformed into E.coli Bl21 plysS and the protein of interest was expressed and subsequently purified using Ni-Sepharose affinity column. The produced scFv antibody was analyzed by SDS-PAGE and western blotting techniques. To assess the binding ability of the produced scFv to TNF-, ELISA experiment was performed. MTT assay was carried out to examine the inhibitory effects of J43 on TNF-α cytotoxicity. Molecular docking of J43 into TNF- trimer was conducted using Z-dock program. Results: The J43 scFv antibody was produced in bacterial expression system. The protein band at 25 kDa on SDS-PAGE was attributed to scFv of interest. The production of scFv antibody was confirmed by using western blotting technique. In ELISA experiment, the produced scFv showed appropriate affinity towards TNF-. However, the identified anti-TNF-α scFv didn’t show any inhibitory activity on TNF-α cytotoxicity. Docking data was analyzed in PIC web site and various interactions of TNF- and J43 were investigated. Conclusion: In the current work, anti-TNF- scFv J43 antibody was expressed in a prokaryotic system and the affinity of the purified protein to TNF- was elucidated. The findings in the current study can be valuable in designing and developing new TNF- inhibitors.