Production of Soluble Tumor Necrosis Factor Receptor Type I in Escherichia coli: Optimization of the Refolding Yields by a Microtiter Dilution Assay
| Autor: | Giovanni Cassani, Silvia Merli, Angelo Corti |
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| Přispěvatelé: | Merli, S, Corti, Angelo, Cassani, G. |
| Rok vydání: | 1995 |
| Předmět: |
Tris
Protein Folding medicine.drug_class Recombinant Fusion Proteins Molecular Sequence Data Biophysics Indicator Dilution Techniques Enzyme-Linked Immunosorbent Assay Monoclonal antibody medicine.disease_cause Biochemistry Receptors Tumor Necrosis Factor chemistry.chemical_compound Affinity chromatography Escherichia coli medicine Animals Bioassay Cloning Molecular Bovine serum albumin Molecular Biology Chromatography Base Sequence biology Dilution assay Microchemistry Titrimetry Cell Biology Solubility chemistry biology.protein Urea Cattle |
| Zdroj: | Analytical Biochemistry. 230:85-91 |
| ISSN: | 0003-2697 |
| DOI: | 10.1006/abio.1995.1441 |
| Popis: | 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 mu g/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. (C) 1995 Academic Press, Inc. |
| Databáze: | OpenAIRE |
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