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 journalArticlepeer-review

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

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Molecular Biology

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