title: Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose.
abstract: OBJECTIVE
  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%.
  
  
  CONCLUSIONS
  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.
authors: Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran. ; Student' Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran.
authors: Jalal
authors: J
authors: Abdolalizadeh
authors: Jafar
authors: J
authors: Majidi Zolbanin
authors: Mohammad
authors: M
authors: Nouri
authors: Behzad
authors: B
authors: Baradaran
authors: Aliakbar
authors: A
authors: Movassaghpour
authors: Safar
authors: S
authors: Farajnia
authors: Yadollah
authors: Y
authors: Omidi
keywords: Affinity Purification; LPS; Monoclonal antibody; TNF-α expression