title: Optimization of TNF-α overexpression in Escherichia coli using response surface methodology: Purification of the protein and oligomerization studies.
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.
authors.affiliation: The Centre for Research and Technology of Thessaly, 3rd km Karditsa-Mitropolis, Karditsa 43100, Greece.
authors.forename: Christos P
authors.initials: CP
authors.lastname: Papaneophytou
authors.forename: George A
authors.initials: GA
authors.lastname: Kontopidis
mesh_terms: D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000737:chemistry; D011136:Polysorbates / Q000737:chemistry; D055503:Protein Multimerization; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012965:Sodium Chloride / Q000737:chemistry; D012995:Solubility; D013696:Temperature; D013997:Time Factors; D017186:Titrimetry / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000302:isolation & purification*; D014764:Viral Proteins / Q000737:chemistry
chemical_list: D011136:Polysorbates; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D012965:Sodium Chloride; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D005990:Glycerol