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

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 (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.

Functional Analysis of the Autographa californica Multiple Nucleopolyhedrovirus GP64 Terminal Fusion Loops and Interactions with Membranes

“…Promiscuous cellular entry by AcMNPV BV, combined with a number of prior studies (10,18,19,43), suggested the possibility that GP64 receptor binding may result from a direct interaction or recognition of phospholipids in the host cell membrane. Therefore, we examined the interactions of purified virions and a purified soluble GP64 protein with artificial membranes that have different phospholipid compositions.…”

Purification of functional baculovirus particles from silkworm larval hemolymph and their use as nanoparticles for the detection of human prorenin receptor (PRR) binding

“…Some transmembrane proteins are displayed on the baculovirus surface without any fusion with the baculovirus envelope protein [15,16,26]. Baculoviruses that display transmembrane proteins on their surfaces are suitable for use in ELISAs, for the expression cloning of a gene from a cDNA library and for making proteoliposomes [17,27]. In particular, baculovirus particles can be used as probes for the expression cloning of receptors and ligands from cDNA libraries using magnetic sorting and flowcytometric analysis [17].…”

Preparation of connexin43‐integrated giant Liposomes by a baculovirus expression–liposome fusion method

“…Akiyoshi group reported that the expressed membrane protein was directly incorporated into the liposome membrane upon in vitro synthesis, leading to pure membrane protein-containing liposomes (Kaneda et al, 2009;Nomura et al, 2008). Yoshimura group proposed a baculovirus expression-liposome fusion method for preparation of proteoliposomes (Fukushima et al, 2008;Tsumoto and Yoshimura, 2009). The membrane fusion phenomena between liposomes and glycoprotein 64 (gp64) displayed on recombinant budded viruses (BVs) of baculovirus (Autographa californica nuclear polyhedrosis viruses, AcNPV) under acidic conditions were applied (Blissard and Wenz, 1992;Oomens and Wertz, 2004;Volkman and Goldsmith, 1985;Wang et al, 1997;Zhang et al, 2003).…”

“…The membrane fusion phenomena between liposomes and glycoprotein 64 (gp64) displayed on recombinant budded viruses (BVs) of baculovirus (Autographa californica nuclear polyhedrosis viruses, AcNPV) under acidic conditions were applied (Blissard and Wenz, 1992;Oomens and Wertz, 2004;Volkman and Goldsmith, 1985;Wang et al, 1997;Zhang et al, 2003). The human thyroid-stimulating hormone receptor (TSHR), with seven membrane-spanning domains as G protein-coupled receptors (GPCRs), and the human nicotinic acetylcholine receptor a-subunit (AChRa) were successfully prepared by this method (Fukushima et al, 2008;Fukushima et al,2009;Tsumoto and Yoshimura, 2009). The advantage of this fusion method is the retention of the original orientation and conformation of the membrane proteins after their incorporation into the liposome.…”