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

Christos P. Papaneophytou, George A. Kontopidis

Research output: Contribution to journalArticle

30 Citations (Scopus)

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: OD600nm prior induction 0.55, a post induction temperature of 25 °C, an IPTG concentration of 1 mM and a post-induction time of 4 h. We have also studied TNF-α oligomerization, the major property of this protein, and a Kd 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.

Original languageEnglish
Pages (from-to)35-44
Number of pages10
JournalProtein Expression and Purification
Volume86
Issue number1
DOIs
Publication statusPublished - Nov 2012

Keywords

  • Induction conditions
  • Oligomerization
  • Response surface methodology
  • Tumor necrosis factor-alpha

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