Title:A Simple and Rapid Method for Expression and Purification of Functional TNF-α Using GST Fusion System
Volume: 16
Issue: 8
Author(s): Ali A. Alizadeh, Maryam Hamzeh-Mivehroud, Malak Farajzadeh, Ali A. Moosavi-Movahedi and Siavoush Dastmalchi
Affiliation:
Keywords:
CD spectropolarimetry, chromatography, GST fusion, MTT assay, protein purification, TNF-α.
Abstract: Tumor necrosis factor alpha (TNF-α) is an inflammatory cytokine, involved in both physiological
and pathological pathways. Although there have been various attempts to express and purify
human TNF-α, the current work introduces a simple, rapid, and efficient method for its production
without loss of biological activity. The protein was expressed based on GST-tagged fusion system in
Escherichia coli under optimized condition. The expressed GST fusion protein was applied to glutathione
affinity column and then, TNF-α was cleaved off the GST using thrombin protease. The purity
of the product was more than 95% and further size exclusion chromatography slightly improved the purity. The purified
human TNF-α was tested for its biological activity and structural analysis, using MTT assay (EC50 of 4.1 ×10E–12 M in
L929 cell death assay) and circular dichroism spectropolarimetry, respectively. The results showed that the method used
in this study enables successful production of highly purified and fully functional TNF-α.
