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

Author(s): Ali A. Alizadeh, Maryam Hamzeh-Mivehroud, Malak Farajzadeh, Ali A. Moosavi-Movahedi, Siavoush Dastmalchi

Journal Name: Current Pharmaceutical Biotechnology

Volume 16 , Issue 8 , 2015

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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.

Keywords: CD spectropolarimetry, chromatography, GST fusion, MTT assay, protein purification, TNF-α.

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Article Details

VOLUME: 16
ISSUE: 8
Year: 2015
Published on: 06 June, 2015
Page: [707 - 715]
Pages: 9
DOI: 10.2174/138920101608150603152549
Price: $65

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