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J. Biol. Chem., Vol. 282, Issue 23, 17289-17296, June 8, 2007

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Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment^*

Hajime Mori¹, Chisa Shukunami^¶||1, Akiko Furuyama, Hiroyuki Notsu, Yuriko Nishizaki^¶, and Yuji Hiraki^¶||2

From the Insect Biomedical Research Center, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585, Protein Crystal Corporation, 2-3-8 Honmachi, Chuo-ku, Osaka 541-0053, the ^¶Department of Cellular Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, and ^||Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

Received for publication, August 23, 2006 , and in revised form, February 20, 2007.

^* This study was supported by a grant from Core Research for Evolutional Science and Technology, the Japan Science and Technology Corporation, and by grants-in-aid from the Ministry of Education, Culture, Sport, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Cellular Differentiation, Inst. for Frontier Medical Sciences, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan. Tel. and Fax: 81-75-751-4633; E-mail: hiraki{at}frontier.kyoto-u.ac.jp.

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