Human Fc gamma receptor IIb expressed in Escherichia coli reveals IgG binding capability

Fc gamma receptors (Fc gamma R) are expressed on immunologically active cel ls where they trigger B and T cell responses and are responsible for the cl earance of immunocomplexes. They occur as type I transmembrane proteins and also in soluble forms (sFcR) comprising only the ecto domains of the recep tors. State-of-the-art research has generated demand for highly pure and ho mogeneous sFc gamma R preparations: first, studies of the immunoregulative potential of the soluble Fc gamma Rs have been hampered by cc-purified grow th factors. Second, they are needed for crystallographic analyses to solve questions such as the exact location of the binding site for IgG on the rec eptor, and the graded affinities of the receptors for different IgG subclas ses. This has been unsuccessful due to limitations in availability and homo geneity of sFc gamma R expressed in eukaryotic cells. In order to address t hese problems we expressed the extracellular part of the human Fc-gamma Rll b 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 sequ encing. The unglycosylated Fc gamma Rllb is active because it binds immobil ized antibody as well as the IgG Fc-fragment in solution. Finally, the rece ptor was crystallized in orthorhombic, tetragonal and hexagonal crystal for ms that diffracted X-rays to resolutions of 1.7 Angstrom, 2.7 Angstrom and 3.8 Angstrom respectively.