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Papaneophytou C.P.,Center For Research And Technology Of Thessaly Cereteth | Kontopidis G.A.,Center For Research And Technology Of Thessaly Cereteth | Kontopidis G.A.,University of Thessaly
Protein Expression and Purification | Year: 2012

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. © 2012 Elsevier Inc. All rights reserved. Source

Papaneophytou C.P.,Center For Research And Technology Of Thessaly Cereteth | Mettou A.K.,Center For Research And Technology Of Thessaly Cereteth | Mettou A.K.,University of Thessaly | Rinotas V.,Agricultural University of Athens | And 5 more authors.
ACS Medicinal Chemistry Letters | Year: 2013

Many active compounds may be excluded from biological assays due to their low aqueous solubility. In this study, a simple method for the determination of the solubility of compounds containing aromatic rings is proposed. In addition to DMSO, five organic solvents for screening experiments of TNF-α inhibitors were explored. DMSO and PEG3350 were the most suitable for both protein stability and ligand-binding experiments. In addition, glycerol is a promising solvent for the screening of other compounds for which it might provide acceptable solubilization, due to its strong tendency to preserve the protein. Moreover, a fluorescence binding assay was developed using the TNF-α/SPD304 system, and a Kd of 5.36 ± 0.21 μM was determined. The results of this study could be used for the future screening of potential TNF-α inhibitors, while the protocols developed in this work could be applied to other proteins. © 2012 American Chemical Society. Source

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