Bacterially expressed human FcγRIIb is soluble and functionally active after in vitro refolding

István Kurucz, Ágnes Hilbert, Attila Kapus, Dávid Medgyesi, G. Koncz, G. Sármay, A. Erdei, J. Gergely

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

A recombinant soluble form of the human Fcγ receptor was produced by engineering a cDNA construct containing the extracellular part of the mature protein. After expression in bacteria as inclusion body, the polypeptide was highly purified and was refolded in vitro with a method that was developed for the renaturation of immunoglobulin fragments. With this method oxidation of the disulfide bridges within the domains of the protein is done in the presence of an artificial 'chaperone' which protects the polypeptide molecules from unwanted protein-protein interactions thereby inhibiting the incorrect oxidation of the SH-groups, and misfolding of the protein. The refolded recombinant soluble FcγRIIb showed several characteristics of the native receptor: (i) it was recognized by a series of monoclonal antibodies specific for, and in most cases produced against the native cell-surface receptor; (ii) it is bound to its ligand (the Fc-region of different immunoglobulins) under very diverse conditions; and (iii) it is competed strongly and specifically with the native cell surface receptor for both ligand and antibody binding in experiments with distinct read-outs; (iv) monoclonal antibodies produced against the recombinant protein specifically recognized FcγRIIb on different cells. From these data it was concluded that the recombinant soluble Fc-receptor was in a native, functionally active form, and its function was not affected by the lack of glycosylation.

Original languageEnglish
Pages (from-to)33-40
Number of pages8
JournalImmunology Letters
Volume75
Issue number1
DOIs
Publication statusPublished - Dec 1 2000

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Fc Receptors
Cell Surface Receptors
Proteins
Monoclonal Antibodies
Ligands
Immunoglobulin Fragments
Peptides
Inclusion Bodies
Glycosylation
Recombinant Proteins
Disulfides
Immunoglobulins
Complementary DNA
Bacteria
In Vitro Techniques
Antibodies
Protein Domains

Keywords

  • Bacterial expression
  • Refolding
  • Soluble Fcγ receptors

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

Bacterially expressed human FcγRIIb is soluble and functionally active after in vitro refolding. / Kurucz, István; Hilbert, Ágnes; Kapus, Attila; Medgyesi, Dávid; Koncz, G.; Sármay, G.; Erdei, A.; Gergely, J.

In: Immunology Letters, Vol. 75, No. 1, 01.12.2000, p. 33-40.

Research output: Contribution to journalArticle

Kurucz, István ; Hilbert, Ágnes ; Kapus, Attila ; Medgyesi, Dávid ; Koncz, G. ; Sármay, G. ; Erdei, A. ; Gergely, J. / Bacterially expressed human FcγRIIb is soluble and functionally active after in vitro refolding. In: Immunology Letters. 2000 ; Vol. 75, No. 1. pp. 33-40.
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