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Journal of Biochemistry Advance Access originally published online on October 4, 2008
Journal of Biochemistry 2008 144(6):763-770; doi:10.1093/jb/mvn125
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© The Authors 2008. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Development of a Novel Preparation Method of Recombinant Proteoliposomes Using Baculovirus Gene Expression Systems

Hidetaka Fukushima1,2, Masashi Mizutani2, Koji Imamura3, Kazuhiko Morino3, Jun Kobayashi4, Katsuzumi Okumura1, Kanta Tsumoto2,5 and Tetsuro Yoshimura2,5,*

1Graduate School of Bioresources; 2Graduate School of Engineering, Mie University, Tsu, Mie 8507; 3Medical and Biological Laboratories Co. Ltd., Ina, Nagano 396-0002; 4Faculty of Agriculture, Yamaguchi University, Yamaguchi, Yamaguchi 753-8515; and 5Liposome Engineering Laboratory, Inc., Tsu, Mie 8507, Japan

*To whom correspondence should be addressed. Tel: +81-(0)59-231-5326, Fax: +81-(0)59-231-5328, E-mail: tyoshi{at}eng.mie-u.ac.jp

Received June 28, 2008; Accepted September 21, 2008


  Abstract

We have developed a novel method for the preparation of ‘recombinant proteoliposomes’. Membrane proteins were expressed on budded virus (BV) envelopes using baculovirus gene expression systems, and proteoliposomes were prepared by fusion of these viruses with liposomes. First, plasmid DNA containing the gene for the thyroid-stimulating hormone receptor (TSHR) or the acetylcholine receptor {alpha}-subunit (AChR{alpha}) was co-transfected with wild type virus [Autographa californica nuclear polyhedrosis virus (AcNPV)] genomes into insect cells [Spodoptera frugiperda (Sf9)] to obtain recombinant viruses via homologous recombination. The recombinant viruses were again infected into Sf9 cells, and the resulting BVs were shown to express TSHR and AChR{alpha}. Next, the fusion behaviour of AcNPV-derived BVs and liposomes was examined via a fluorescence assay, and BVs were shown to fuse with phosphatidylserine-containing liposomes below pH 5.0, the pH at which fusion glycoprotein gp64 on the virus envelope becomes active. TSHR- or AChR{alpha}-expressed BVs were also shown to fuse with liposomes. Finally, TSHR- and AChR{alpha}-recombinant proteoliposomes were immobilized on enzyme-linked immunosorbent assay plates, and their reactivities were examined via a general immunoassay, which showed that the recombinant proteoliposomes were fully active. These results successfully demonstrate the development of a method based on a baculovirus gene expression system for the preparation of recombinant and functional proteoliposomes.

Key Words: baculovirus, gene expression, membrane fusion, membrane receptor, proteoliposome

Abbreviations: AChR, acetylcholine receptor; AChR{alpha}, acetylcholine receptor {alpha}-subunit; AcNPV, Autographa californica nuclear polyhedrosis virus; Bt-PE, 1,2-dipalmitoyl-sn-glycero-3-(N-biotinyl) phosphoethanolamine; BV, budded virus; ELISA, enzyme-linked immunosorbent assay; IFV, influenza virus; LUV, large unilamellar vesicle; MCS, multiple cloning site; MLV, multilamellar vesicle; PA, phosphatidic acid; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; R18, octadecyl rhodamine B chloride; Sf9, Spodoptera frugiperda; TAE, Tris–Acetate-EDTA; TSHR, thyroid-stimulating hormone receptor


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