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Optimization of TNF-alpha, overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies
dc.creator | Papaneophytou, C. P. | en |
dc.creator | Kontopidis, G. A. | en |
dc.date.accessioned | 2015-11-23T10:43:58Z | |
dc.date.available | 2015-11-23T10:43:58Z | |
dc.date.issued | 2012 | |
dc.identifier | 10.1016/j.pep.2012.09.002 | |
dc.identifier.issn | 1046-5928 | |
dc.identifier.uri | http://hdl.handle.net/11615/31866 | |
dc.description.abstract | Tumor necrosis factor-alpha (TNF-alpha) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-alpha is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-alpha inhibitors. In this work, induction conditions for the production of human TNF-alpha fusion protein in a soluble form by recombinant Escherichia coli BL21(DE3) pLysS were optimized using response surface methodology based on the central composite design. The induction conditions included cell density prior induction (OD600nm), post-induction temperature, IPTG concentration and post-induction time. Statistical analysis of the results revealed that all variables and their interactions had significant impact on production of soluble TNF-alpha. An 11% increase of TNF-alpha production was achieved after determination of the optimum induction conditions: OD600nm prior induction 0.55, a post induction temperature of 25 degrees C, an IPTG concentration of 1 mM and a post-induction time of 4 h. We have also studied TNF-alpha oligomerization, the major property of this protein, and a K-d value of 0.26 nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable Kd value for protein trimerization probably due to the complexibility of our model. (C) 2012 Elsevier Inc. All rights reserved. | en |
dc.source | Protein Expression and Purification | en |
dc.source.uri | <Go to ISI>://WOS:000310654600006 | |
dc.subject | Tumor necrosis factor-alpha | en |
dc.subject | Induction conditions | en |
dc.subject | Response surface | en |
dc.subject | methodology | en |
dc.subject | Oligomerization | en |
dc.subject | TUMOR-NECROSIS-FACTOR | en |
dc.subject | INCLUSION-BODY PROTEINS | en |
dc.subject | RECOMBINANT PROTEINS | en |
dc.subject | SOLUBLE-PROTEIN | en |
dc.subject | EXPRESSION | en |
dc.subject | CULTURE | en |
dc.subject | ARTHRITIS | en |
dc.subject | PROMOTER | en |
dc.subject | Biochemical Research Methods | en |
dc.subject | Biochemistry & Molecular Biology | en |
dc.subject | Biotechnology & Applied Microbiology | en |
dc.title | Optimization of TNF-alpha, overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies | en |
dc.type | journalArticle | en |
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