CD64, also known as the type I Fc-gamma receptor, functions as a high affinity receptor for IgG. It is involved in the activation of phagocytosis, endocytosis of IgG-opsonized particles, and activation of the complement system. Our goal is to determine the structure of CD64 and its complex with immunoglobulins. To express and purify the recombinant CD64 protein, we have constructed both bacteria and a CHO cell based expression system. The yield of the CHO cell expression remained at a level of 50 ug/liter of cell culture after gene amplification. The bacteria system, on the other hand, yielded the protein in an inactive inclusion body form. Subsequent refolding experiments resulted in less than a mg of the protein for crystallization. Currently, we are attempting to express the recombinant CD64 using a baculovirus expression system. B cells initiate their signaling through the formation of antigen cross-linked B cell receptor (BCR) which consists of an immunoglobulin (Ig) molecule and Ig alpha and Ig beta. In order to study the molecular mechanism of BCR activation, we propose to determine the crystal structure of the BCR Ig, Ig alpha and Ig beta complex. We have constructed bacteria expression systems to express both the human Ig alpha and Ig beta. Large quantities of Ig alpha and Ig beta inclusion bodies have been obtained using these expression systems. We are currently attempting to refold Ig alpha and Ig beta to reconstitute in vitro a BCR complex for crystallization and structure determination.
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| Radaev, Sergei; Li, Sean; Sun, Peter D (2006) A survey of protein-protein complex crystallizations. Acta Crystallogr D Biol Crystallogr 62:605-12 |
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| Radaev, S; Sun, P D (2001) Recognition of IgG by Fcgamma receptor. The role of Fc glycosylation and the binding of peptide inhibitors. J Biol Chem 276:16478-83 |
| Zhang, Y; Boesen, C C; Radaev, S et al. (2000) Crystal structure of the extracellular domain of a human Fc gamma RIII. Immunity 13:387-95 |
