Fabrication and Application of Protein Crystal Microarrays
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Fabrication and Application of Protein Crystal Microarrays Yoichiroh Hosokawa, Satoshi Matsumura, Hiroshi Y. Yoshikawa, Hiroshi Masuhara *, Ryosuke Nakamura1, Yasuo Kanematsu1, Keiko Ikeda2, Ai Shimo-oka3, and Hajime Mori2,3 Department of Applied Physics, Handai Frontier Research Center, and Venture Business Laboratory, Osaka University, Suita, Osaka 565-0871, Japan 1 Venture Business Laboratory, Osaka University, Suita, Osaka 565-0871, Japan 2 Protein Crystal Corporation, Honmachibashi-nishi Building, 1-1-3, Honmachi, Chuo-ku, Osaka 541-0053, Japan 3 Department of Applied Biology, Faculty of Textile Science, Kyoto Institute of Technology, Matsugasaki, Sakyo-ku, Kyoto 606-8585 *
Corresponding author. Tel: +81-6-6879-7837, Fax: +81-6-6876-8580 e-mail: [email protected]
ABSTRACT With the aim of fabricating highly integrated protein microarrays, functional proteins were occluded into a protein crystal, consisting of a proteinaceous occlusion body termed polyhedrin, and the crystals were individually fixed onto a polymer film by using laser trapping and bonding techniques. Individual crystals were trapped by the 1064 nm beam of a Nd3+:YAG laser and placed to the film. The bonding to the film was achieved by irradiating with the single shot of the 800 nm beam of a femtosecond Ti:sapphire laser. Enhanced green fluorescent protein (EGFP) was occluded into the polyhedrin crystal and the crystals were patterned and observed by florescence imaging and single crystal florescence spectroscopy. From these experiments, the biological activity of the bonded crystal was confirmed and their potential as element in protein microarrays was discussed.
INTRODUCTION Some insect virus infections result in the production of a number of large protein crystals (occlusion bodies), termed polyhedra, and many virus particles are occluded within the polyhedra. Recently, some of the present authors have successfully demonstrated that various functional proteins can be immobilized into the polyhedra by using gene manipulation techniques [1-3]. Some of the proteins occluded into the polyhedra will be on the surface of the crystal and some of the active sites will be exposed rather than buried. Therefore they will be able to interact with other proteins or chemicals in solution, as illustrated in figure 1 (A). We have already conducted
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development of a micro fixation technique by using a laser trapping technique based on the photon pressure of a focused near infrared laser beam [4-7]. When µm-sized crystals are manipulated one by one without damage, bonded on the substrate, and installed into a microchip, highly integrated protein microarrays will be achieved. In this paper, a method to fabricate protein microarrays is proposed and demonstrated. Furthermore, it was confirmed by means of enhanced green fluorescent protein (EGFP) occluded into the crystal that there was no damages to their biological activity. The fluorescence spectra before and after fixation were investigated by fluorescence imaging and fluorescence
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