Ali Akbar Alizadeh1, Maryam Hamzeh-Mivehroud2, Siavoush Dastmalchi3. 1. Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Students Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 2. Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. 3. Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. ; School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
Abstract
PURPOSE: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. METHODS: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. RESULTS: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. CONCLUSION: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality.
PURPOSE: TNF-α is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-α antibodies are being used in clinic for the purpose of diagnosis and treatment due to their high specificity. The objective of the current study was to express and purify an anti-TNF-α scFv antibody identified by phage display technology. METHODS: The DNA coding sequence of the identified scFv was cloned into pET28a vector and the corresponding protein was expressed as 6×His tagged using E.coli BL21 (DE3) pLysS expression system followed by affinity purification on Ni-Sepharose affinity column. RESULTS: The J44 scFv antibody was cloned into the expression vector and successfully expressed and purified. The purity of the scFv fraction was confirmed using SDS-PAGE analysis. Western blotting technique was used to detect expression of 6×His tagged protein. CONCLUSION: In the current study an anti-TNF-α scFv antibody was successfully expressed in bacterial expression system and purified on affinity column. The purified protein can be used in different in vitro and in vivo experiments in order to elucidate its functionality.
Entities:
Keywords:
Affinity purification; Gene cloning; Protein expression; TNF-α