A Simple and Rapid Method for Expression and Purification of Functional TNF-α Using GST Fusion System

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.

Cite this article as:

Alizadeh A. Ali, Hamzeh-Mivehroud Maryam, Farajzadeh Malak, Moosavi-Movahedi A. Ali and Dastmalchi Siavoush, A Simple and Rapid Method for Expression and Purification of Functional TNF-α Using GST Fusion System, Current Pharmaceutical Biotechnology 2015; 16 (8) . https://dx.doi.org/10.2174/138920101608150603152549