title: A Simple and Rapid Method for Expression and Purification of Functional TNF-α Using GST Fusion System.
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 (EC(50) 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.
authors.forename: Ali A
authors.initials: AA
authors.lastname: Alizadeh
authors.forename: Maryam
authors.initials: M
authors.lastname: Hamzeh-Mivehroud
authors.forename: Malak
authors.initials: M
authors.lastname: Farajzadeh
authors.forename: Ali A
authors.initials: AA
authors.lastname: Moosavi-Movahedi
authors.affiliation: School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran. dasmalchi.s@tbzmed.ac.ir.
authors.forename: Siavoush
authors.initials: S
authors.lastname: Dastmalchi
mesh_terms: D016923:Cell Death; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C439524:TNF protein, human; D014409:Tumor Necrosis Factor-alpha; D005982:Glutathione Transferase