Production, purification and binding ability of anti-FGF7 sdAb D53 antibody identified by phage display technique

Abstract

Introduction: Fibroblast growth factor 7 (FGF7) is a member of the fibroblast growth factor (FGF) family. FGFs are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth. Therefore, inhibition of FGF7 can be an effective treatment for such pathological diseases. Aims: In this study, we aimed to investigate the production, purification and binding ability of a single domain anti-FGF7 antibody (i.e., D53) identified by phage display technique against FGF7. Material and Methods: The present stop codon in CDR2 region of heavy variable chain was converted to glutamine codon with site directed mutagenesis and then the corrected sequence was cloned into pGEX-6p-1 expression vector. The constructed vector was transformed into E.coli origami and the protein of interest was expressed and subsequently purified using Glutathione-Sepharose affinity column. The produced domain antibody was analyzed by SDS-PAGE and western blotting techniques. To assess the binding ability of the produced antibody toFGF7, ELISA experiment was performed. Molecular docking study was carried out to investigate the mode of interaction of D53 with FGF7. Results: The D53 domain antibody was produced fused to GST tag in bacterial expression system. The protein band at about 40 kDa on SDS-PAGE was attributed to dAb of interest attached to GST. The production of D53 domain antibody was confirmed by using western blotting technique. ELISA experiment showed that the produced sdAb is able to bind FGF7 with Kd value of 0.6±0.1 μM. Molecular docking study followed by protein interaction calculator (PIC) revealed that most of the interactive residues of D53 are located at CDR3. Conclusion: In the current work, anti-FGF7 sdAb D53 antibody was expressed in a prokaryotic system and its affinity towards FGF7 was elucidated. The findings in the current study can be valuable in designing and developing new FGF7 inhibitors