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.
In this study, we aimed to investigate the production, purification and binding ability of single domain anti-FGF7 antibody (i.e., D53) identified by phage display technique against FGF7.
Methods: The DNA sequence of D53 antibody was modified to change stop codon present in CDR2 region of heavy variable chain 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 of D53 into FGF7 was conducted using Z-dock program.
Results: The D53 domain antibody was produced in bacterial expression system. The protein band at about 13 kDa on SDS-PAGE was attributed to sdAb of interest. The production of D53 domain antibody was confirmed by using western blotting technique. In ELISA experiment, the produced sdAb showed appropriate affinity towards FGF7. Docking data was analyzed in protein interaction calculator (PIC) web site and various interactions of FGF7 and D53 were investigated.
Conclusion: In the current work, anti-FGF7 sdAb D53 antibody was expressed in a prokaryotic system and the affinity of the purified protein was elucidated. The findings in the current study can be valuable in designing and developing new FGF7 inhibitors.
Keywords: CAF , FGF7, sdAb, Affinity Chromatography, Westorn blot