Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled
Jalal Abdolalizadeh1, Jafar Majidi Zolbanin, Mohammad Nouri
1Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran. ; Student' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
Purpose:
Recombinant tumor necrosis factor-alpha (TNF-α) 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-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α 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-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α 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-α protein can be applied for various in vitro and in vivo applications.
Insights
This study demonstrates the effectiveness of anti-tumor necrosis factor-alpha (TNF-α) single-chain variable fragments (scFv) for purifying recombinant TNF-α. The developed method achieves high purity, enabling potential therapeutic applications.
Area of Science:
- Biotechnology
- Immunology
- Protein Chemistry
Background:
- Recombinant tumor necrosis factor-alpha (TNF-α) is an antineoplastic agent used for melanoma and sarcoma.
- TNF-α targets tumor cells by affecting tumor-associated vessels.
- Affinity chromatography is a standard method for pharmaceutical protein purification.
Purpose of the Study:
- To evaluate anti-TNF-α single-chain variable fragments (scFv) for purifying TNF-α.
- To assess the purity of TNF-α obtained using scFv-based affinity chromatography.
Main Methods:
- Raji cells were induced to express TNF-α using lipopolysaccharides (LPS).
- TNF-α expression was confirmed by Western blotting and FACS flow cytometry.
- Anti-TNF-α scFv, selected from a phage display library, were coupled to CNBr-activated sepharose 4B beads for affinity purification.
- Protein purity was analyzed using SDS-PAGE.
Main Results:
- Successful expression of TNF-α in Raji cells was confirmed.
- SDS-PAGE analysis demonstrated that the scFv effectively purified TNF-α to over 95% purity.
Conclusions:
- The produced scFv antibody fragments show significant potential for TNF-α purification.
- Highly pure recombinant TNF-α obtained can be utilized in various in vitro and in vivo applications.
