Human Fcgamma receptor IIb expressed in Escherichia coli reveals IgG binding capability.
Fcgamma receptors (FcgammaR) are expressed on immunologically active cells where they trigger B and T cell responses and are responsible for the clearance of immunocomplexes. They occur as type I transmembrane proteins and also in soluble forms (sFcR) comprising only the ecto domains of the receptors. State-of-the-art research has generated demand for highly pure and homogeneous sFcgammaR preparations: first, studies of the immunoregulative potential of the soluble FcgammaRs have been hampered by co-purified growth factors. Second, they are needed for crystallographic analyses to solve questions such as the exact location of the binding site for IgG on the receptor, and the graded affinities of the receptors for different IgG subclasses. This has been unsuccessful due to limitations in availability and homogeneity of sFcgammaR expressed in eukaryotic cells. In order to address these problems we expressed the extracellular part of the human FcgammaRIIb in E. coli. The protein was refolded, purified in a three-step procedure and characterized by SDS-PAGE, mass spectrometry as well as N-terminal sequencing. The unglycosylated FcgammaRIIb is active because it binds immobilized antibody as well as the IgG Fc-fragment in solution. Finally, the receptor was crystallized in orthorhombic, tetragonal and hexagonal crystal forms that diffracted X-rays to resolutions of 1.7 A, 2.7 A and 3.8 A respectively.
Sondermann
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P
Max-Planck-Institut für Biochemie, Abteilung Strukturforschung, Martinsried, Germany.
Jacob
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U
D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002850:Chromatography, Gel; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism*; D011485:Protein Binding; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D015703:Antigens, CD; D017931:DNA Primers; C080571:Fc gamma receptor IIB; D007074:Immunoglobulin G; D017452:Receptors, IgG; D011994:Recombinant Proteins