Affinity Purification of Tumor Necrosis Factor-汐 Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

作者：Jalal Abdolalizadeh; Jafar Majidi Zolbanin; Mohammad Nouri; Behzad Baradaran

来源：Advanced Pharmaceutical Bulletin, 2013.

DOI：10.5681/apb.2013.004

• TNF-&alpha
• expression
• Affinity Purification
• Monoclonal antibody
• LPS

Purpose: Recombinant tumor necrosis factor-alpha (TNF-汐) 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-汐 produced by Raji cells. he Raji cells were induced by lipopolysaccharides (LPS) to express TNF-汐. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-汐 expression. The anti-TNF-汐 scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-汐 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-汐 with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-汐 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-汐 protein can be applied for various in vitro and in vivo applications.