Affinity purification of tumor necrosis factor-? expressed in raji cells by produced scFv antibody coupled CNBr-activated sepharose

Abstract

Purpose: Recombinant tumor necrosis factor-alpha (TNF-?) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods: In this study, we examined the potential of our produced anti-TNF-??scFv fragments for purification of TNF-? produced by Raji cells. ??he Raji cells were induced by lipopolysaccharides (LPS) to express TNF-?. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-? expression. The anti-TNF-? scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-? and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-? with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-? protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-? protein can be applied for various in vitro and in vivo applications. آ© 2013 by Tabriz University of Medical Sciences.