Crystallization of secreted protein by complexation with an antibody fragment

Taro Tamada,^a Keiko Kurosawa,^a Uichi Nishiyama,^b Tomoaki Kuwaki,^b and Ryota Kuroki^a

^aPharmaceutical Research Laboratories, Pharmaceutical Division, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa 236-0004, Japan; ^bPharmaceutical Development Laboratory, Pharmaceutical Division, Kirin Brewery Co., Ltd., 3 Miyahara-cho, Takasaki 370-1295, Japan (ttamada@kirin.co.jp)

It is known that secreted glycoproteins, such as cytokines, are hard to crystallize in spite of their industrial importance. Our approach for crystallization of these proteins is the use of antibody fragment (Fab). The first example was the crystallization of thrombopoietin (TPO). TPO is a cytokine which primarily stimulates megakaryocytopoiesis and thrombopoiesis. TPO was crystallized by complexation with Fab derived from a neutralizing monoclonal antibody [1]. The tertiary structure of TPO was also successfully determined only by molecular replacement technique using the known coordinates of Fab as a search model. It was confirmed that the use of Fab was effective not only for protein crystallization, but also for phase determination.

The second example is the crystallization of soluble receptors. The extracellular domain of receptors is the entrance of signals induced by ligand binding. The interaction of the receptors with several drug candidates is widely investigated. To elucidate activation mechanism of TPO receptor by ligand binding, we aim to determine the complex structure of TPO and its receptor by complexation with Fab. The extracellular region of human TPOR expressed using by the animal cell was used for crystallization. Several Fab fragments derived from a monoclonal antibody that recognize the extracellular domain of TPOR were used for the search of crystallization condition. After various screening for crystallization condition, TPO/TPOR/Fab complex, and TPOR/Fab complex, were successfully obtained with the use of one of the Fab fragments. Although further optimization of crystallization condition is needed, the use of Fab was effective approach for TPOR crystallization. The structure information of TPOR will give the useful information for pharmaceutical development of TPO.

References

1 Kuroki, R., Hirose, M., Kato, Y., Feese, M. D., Tamada, T., Shigematsu, H., Watarai, H., Maeda, Y., Tahara, T., Kato, T., & Miyazaki, H. (2002) Acta Cryst. D58, 856-858.