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.
Current Pharmaceutical Biotechnology
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.
Export Options
About this article
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
DOI https://dx.doi.org/10.2174/138920101608150603152549 |
Print ISSN 1389-2010 |
Publisher Name Bentham Science Publisher |
Online ISSN 1873-4316 |
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
The Protective Effects of Natural Products on Blood-Brain Barrier Breakdown
Current Medicinal Chemistry Neutrophil MiRNA-128-3p is Decreased During Active Phase of Granulomatosis with Polyangiitis
Current Genomics Survey of the State of the Art in Biomaterials, Cells, Genes and Proteins Integrated into Micro- and Nanoscaffolds for Tissue Regeneration
Current Nanoscience Berberine Inhibits the Gene Expression and Production of Proinflammatory Cytokines by Mononuclear Cells in Rheumatoid Arthritis and Healthy Individuals
Current Rheumatology Reviews Sex Steroid Hormones, Cardiovascular Diseases and The Metabolic Syndrome
Cardiovascular & Hematological Agents in Medicinal Chemistry Stroke in Women - Oral Contraception, Pregnancy, and Hormone Replacement Therapy
Current Vascular Pharmacology Retinoids as Critical Modulators of Immune Functions: New Therapeutic Perspectives for Old Compounds
Endocrine, Metabolic & Immune Disorders - Drug Targets Protective Role of Diabetes Mellitus on Abdominal Aortic Aneurysm Pathogenesis: Myth or Reality?
Current Vascular Pharmacology Glucocorticoids and the Cardiovascular System: State of the Art
Current Pharmaceutical Design Interrelationships Among Gut Microbiota and Host: Paradigms, Role in Neurodegenerative Diseases and Future Prospects
CNS & Neurological Disorders - Drug Targets Targeting Potassium Channels: New Advances in Cardiovascular Therapy
Recent Patents on Cardiovascular Drug Discovery Steroid Induced Bilateral Avascular Necrosis of Head of Femur in an Adult Male Patient - A Case Report
Current Drug Safety PDE5 Inhibitors in Non-Urological Conditions
Current Pharmaceutical Design The Anti-Inflammatory Potential of ACE2/Angiotensin-(1-7)/Mas Receptor Axis: Evidence from Basic and Clinical Research
Current Drug Targets Ocular Toxocariasis: New Diagnostic and Therapeutic Perspectives
Recent Patents on Anti-Infective Drug Discovery Heat Shock Proteins - Two Sides of a Coin
Current Cardiology Reviews Nitrofurantoin Pulmonary Toxicity: Neglected Threat
Current Drug Safety Emerging Targets in Neurodegeneration: New Opportunities for Alzheimer's Disease Treatment?
Current Topics in Medicinal Chemistry Noscapine and its Analogs as Chemotherapeutic Agent: Current updates
Current Topics in Medicinal Chemistry Biological Agents in Familial Mediterranean Fever Focusing on Colchicine Resistance and Amyloidosis
Current Medicinal Chemistry