title: Incorporation of adenylate cyclase into membranes of giant liposomes using membrane fusion with recombinant baculovirus-budded virus particles.
abstract: Recombinant transmembrane adenylate cyclase (AC) was incorporated into membranes of giant liposomes using membrane fusion between liposomes and baculovirus-budded virus (BV). AC genes were constructed into transfer vectors in a form fused with fluorescent protein or polyhistidine at the C-terminus. The recombinant BVs were collected by ultracentrifugation and AC expression was verified using western blotting. The BVs and giant liposomes generated using gentle hydration were fused under acidic conditions; the incorporation of AC into giant liposomes was demonstrated by confocal laser scanning microscopy through the emission of fluorescence from their membranes. The AC-expressing BVs were also fused with liposomes containing the substrate (ATP) with/without a specific inhibitor (SQ 22536). An enzyme immunoassay on extracts of the sample demonstrated that cAMP was produced inside the liposomes. This procedure facilitates direct introduction of large transmembrane proteins into artificial membranes without solubilization.
authors.affiliation: Molecular Bioengineering Laboratory, Division of Chemistry for Materials, Graduate School of Engineering, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie, 514-8507, Japan, hawk.fall.mie@gmail.com.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Mori
authors.forename: Koki
authors.initials: K
authors.lastname: Kamiya
authors.forename: Masahiro
authors.initials: M
authors.lastname: Tomita
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Yoshimura
authors.forename: Kanta
authors.initials: K
authors.lastname: Tsumoto
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000201:enzymology* / Q000235:genetics; D000242:Cyclic AMP / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D008561:Membrane Fusion; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000201:enzymology*
chemical_list: D008081:Liposomes; D011994:Recombinant Proteins; D000242:Cyclic AMP; D000262:Adenylyl Cyclases