Development of a novel preparation method of recombinant proteoliposomes using baculovirus gene expression systems.
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 (AChRalpha) 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 AChRalpha. 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 AChRalpha-expressed BVs were also shown to fuse with liposomes. Finally, TSHR- and AChRalpha-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.
Fukushima
Hidetaka
H
Graduate School of Bioresources, Mie University, Tsu, Mie 8507, Japan.
Mizutani
Masashi
M
Imamura
Koji
K
Morino
Kazuhiko
K
Kobayashi
Jun
J
Okumura
Katsuzumi
K
Tsumoto
Kanta
K
Yoshimura
Tetsuro
T
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018411:Spodoptera / Q000378:metabolism; D014162:Transfection; D014760:Viral Fusion Proteins / Q000235:genetics / Q000378:metabolism
D008565:Membrane Proteins; D011510:Proteolipids; D011993:Recombinant Fusion Proteins; D014760:Viral Fusion Proteins; C020485:proteoliposomes