Bacterially expressed human Fc gamma RIIb is soluble and functionally active after in vitro refolding.
A recombinant soluble form of the human Fc gamma 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 Fc gamma RIIb 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 Fc gamma RIIb 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.
Kurucz
I
I
Department of Cellular and Molecular Biology of Biorex R&D Co., Veszprem, Hungary. istvan_kurucz@rex.biorex.hu
Hilbert
A
A
Kapus
A
A
Medgyesi
D
D
Koncz
G
G
Sármay
G
G
Erdei
A
A
Gergely
J
J
D000818:Animals; D000906:Antibodies / Q000276:immunology; D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D001402:B-Lymphocytes; D015153:Blotting, Western; D020013:Calcium Signaling; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D051379:Mice; D008807:Mice, Inbred BALB C; D017510:Protein Folding; D017452:Receptors, IgG / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D000906:Antibodies; D000911:Antibodies, Monoclonal; D015703:Antigens, CD; C080571:Fc gamma receptor IIB; D008024:Ligands; D017452:Receptors, IgG; D011994:Recombinant Proteins