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Advanced Pharmaceutical Bulletin
ISSN: 2228-5881      eISSN: 2251-7308  
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Article History
Submitted: 26 Sep 2012
First published online: 07 Feb 2013

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Adv Pharm Bull. 2013;3(1):19-23 doi: 10.5681/apb.2013.004
PMID:24312807        PMCID:PMC3846060

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

Original Research

Jalal Abdolalizadeh, Jafar Majidi Zolbanin, Mohammad Nouri, Behzad Baradaran, AliAkbar Movassaghpour, Safar Farajnia, Yadollah Omidi *


Abstract
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.





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Articles by Abdolalizadeh J
Articles by Majidi Zolbanin J
Articles by Nouri M
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Articles by Movassaghpour A
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Articles by Abdolalizadeh J
Articles by Majidi Zolbanin J
Articles by Nouri M
Articles by Baradaran B
Articles by Movassaghpour A
Articles by Farajnia S
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