Monday, July 27, 2009
P40
Expression of recombinant soluble human FcγRI with high affinity for immunoglobulin G
Teruhiko Ide, Toru Tanaka, Yoshiharu Asaoka, and Keiichi Murayama. Tokyo Research Center, Tosoh corporation, 2743-1 Hayakawa, Ayase Kanagawa, Ayase-shi, Japan
Human receptors that recognize the Fc portion of IgG are found on most immune system cells and comprise a protein family that is divided into three classes: hFcγRI, hFcγRII, and hFcRγIII based on differences in receptor structure, cell distribution, and affinity for IgG. Human FcγRI is functionally unique as it is the only FcγR able to bind monomeric IgG with high affinity. Since the start of the characterization of the genes for hFcγRI, the production of a functional soluble form of the receptor has been troublesome. So far, yields have been disappointingly low and assays for binding activity to hFcγRI still have to rely on the use of cell lines. Therefore, we have tried to establish a stably transformed CHO cell line expressing the hFcγRI. Production of the hFcγRI was successfully developed with a novel reactor to replace the conventional 2-D cell culturing devices. The His6-tagged recombinant protein was stably secreted into the culture supernatant of CHO cells and recovered using an expanded-bed absorption system. Binding activity to human IgG was verified by ELISA and the isotype-specificity determined by a surface plasmon resonance assay was found to be the same as for native FcγRI. Efficient inhibition was observed for IgG1, IgG3, and IgG4 while no inhibition was observed for IgG2. The hFcγRI strongly bound human IgG1 with a Kd of 0.9 nM. Thus hFcγRI provides a valuable tool for further studying FcγRI structure and function.
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