Production and Purification of a Novel Anti-TNF-alpha Single Chain Fragment Variable Antibody

Abstract

Purpose: TNF-alpha is an inflammatory cytokine with a key role in initiation of inflammatory responses. Anti-TNF-alpha 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-alpha 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 6xHis 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 6xHis tagged protein. Conclusion: In the current study an anti-TNF-alpha 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.