Production of soluble tumor necrosis factor receptor type I in Escherichia coli: optimization of the refolding yields by a microtiter dilution assay.
In this study optimization of the soluble tumor necrosis factor receptor type I (sTNF-RI) refolding by the use of a micro-renaturation assay in 96-well microplates is described. Microplate wells were filled with buffers varying in pH and urea and substrate concentration. Denatured and reduced sTNF-RI was then rapidly diluted and allowed to refold for a variable time at different temperatures. The extent of renaturation was measured by a sandwich enzyme-linked immunosorbent assay (ELISA), based on the use of two monoclonal antibodies obtained against urinary sTNF-RI. Among about 100 different combinations tested, a maximum refolding yield of 21.5% has been obtained in 100 mM Tris, pH 8-8.5, 1 mM EDTA, 0.1% bovine serum albumin, 2 M urea, at a denatured protein concentration of 10 micrograms/ml and at 26 degrees C. Folded sTNF-RI was purified by batchwise immunoaffinity chromatography and its activity evaluated by immunological and biological assays. A good correlation was observed between the data obtained with different assays (biological assay, ligand-directed ELISA, and double-determinant sandwich ELISA) indicating that the refolded receptor has gained biological and immunological reactivity comparable to those of the soluble TNF-receptor type I expressed in eukaryotic cells.
Merli
S
S
Molecular Biology Unit, Tecnogen S.c.p.A, Piana di Monte Verna (CE), Italy.
Corti
A
A
Cassani
G
G
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D007201:Indicator Dilution Techniques; D008832:Microchemistry; D008969:Molecular Sequence Data; D017510:Protein Folding; D018124:Receptors, Tumor Necrosis Factor / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility; D017186:Titrimetry
D018124:Receptors, Tumor Necrosis Factor; D011993:Recombinant Fusion Proteins