Production of soluble tumor necrosis factor receptor type I in Escherichia coli: Optimization of the refolding yields by a microtiter dilution assay

Silvia Merli, Angelo Corti, Giovanni Cassani

Research output: Contribution to journalArticle

Abstract

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 μg/ml and at 26°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.

Original languageEnglish
Pages (from-to)85-91
Number of pages7
JournalAnalytical Biochemistry
Volume230
Issue number1
DOIs
Publication statusPublished - 1995

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Receptors, Tumor Necrosis Factor, Type I
Escherichia coli
Dilution
Assays
Enzyme-Linked Immunosorbent Assay
Immunosorbents
Biological Assay
Urea
Tumor Necrosis Factor Receptors
Eukaryotic Cells
Bovine Serum Albumin
Edetic Acid
Enzymes
Chromatography
Buffers
Monoclonal Antibodies
Ligands
Temperature
Proteins
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

Cite this

Production of soluble tumor necrosis factor receptor type I in Escherichia coli : Optimization of the refolding yields by a microtiter dilution assay. / Merli, Silvia; Corti, Angelo; Cassani, Giovanni.

In: Analytical Biochemistry, Vol. 230, No. 1, 1995, p. 85-91.

Research output: Contribution to journalArticle

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