Recombinant proteoliposomes prepared using baculovirus expression systems.
Proteoliposomes are useful for investigating the functions and properties of membrane proteins. We have established a novel method to prepare proteoliposomes using a baculovirus (Autographa californica nuclear polyhedrosis virus; AcNPV) gene expression system producing the recombinant membrane proteins to be reconstituted. This method consists of two key steps for production of recombinant proteoliposomes: (1) The cDNA of the recombinant membrane protein on a baculovirus vector is transfected into insect cells, and recombinant AcNPV budded viruses (BVs), which express the targeted proteins on their own envelopes, are collected, and (2) the BV envelopes are subjected to fusion with liposomes containing acidic phospholipids by activating the viral glycoprotein gp64 at low pH. In this chapter, we describe the reconstitution of the transmembrane proteins, thyroid-stimulating hormone receptor (TSHR, a member of G protein-coupled receptor family) and acetylcholine receptor alpha-subunit (AChRalpha), in various types of liposomes, including large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs), and giant unilamellar vesicles (GUVs). Enzyme-linked immunosorbent assay (ELISA) shows that the reconstituted proteins (TSHR and AChRalpha) specifically bind their ligands (TSH and alpha-bungarotoxin), respectively, as well as antibodies against the recombinant proteoliposomes. In addition, TSHRs reconstituted on proteo-GUVs are successfully visualized using a confocal laser scanning microscope with fluorescence immunostain. These data suggest that recombinant proteoliposomes prepared using the novel method have the potential to be applied for reconstitution of complicated multicomponent membrane protein systems.
Tsumoto
Kanta
K
Graduate School of Engineering, Mie University, Tsu, Mie, Japan.
Yoshimura
Tetsuro
T
D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008561:Membrane Fusion; D008565:Membrane Proteins / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011510:Proteolipids; D011995:Recombination, Genetic
D008565:Membrane Proteins; D011510:Proteolipids; C020485:proteoliposomes