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Protein Expr Purif. 2012 Nov;86(1):35-44. doi: 10.1016/j.pep.2012.09.002. Epub 2012 Sep 16.

Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies.

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  • 1The Centre for Research and Technology of Thessaly, 3rd km Karditsa-Mitropolis, Karditsa 43100, Greece.

Abstract

Tumor necrosis factor-α (TNF-α) is responsible for many autoimmune disorders including rheumatoid arthritis, psoriasis, Chron's disease, stroke, and atherosclerosis. Thus, inhibition of TNF-α is a major challenge in drug discovery. However, a sufficient amount of purified protein is needed for the in vitro screening of potential TNF-α inhibitors. In this work, induction conditions for the production of human TNF-α 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 (OD(600nm)), 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-α. An 11% increase of TNF-α production was achieved after determination of the optimum induction conditions: OD(600nm) prior induction 0.55, a post induction temperature of 25°C, an IPTG concentration of 1mM and a post-induction time of 4h. We have also studied TNF-α oligomerization, the major property of this protein, and a K(d) value of 0.26nM for protein dimerization was determined. The concentration of where protein trimerization occurred was also detected. However, we failed to determine a reliable K(d) value for protein trimerization probably due to the complexibility of our model.

Copyright © 2012 Elsevier Inc. All rights reserved.

[PubMed - indexed for MEDLINE]
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