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ABSTRACT: Giant liposomes are cell-size artificial vesicles with phospholipid bilayers. Here, in order to obtain giant liposomes containing membrane proteins (giant proteoliposomes), we have introduced a novel procedure using membrane fusion phenomena between liposomes and recombinant baculovirus. We tried to thereby reconstitute heterotrimeric guanine nucleotide-binding protein (G protein) that is a peripheral membrane protein, i.e., Gs protein consisting of alpha<sub>s</sub>, szlig and gamma, in giant proteoliposomes. Western blot analysis showed that expression of these proteins on both recombinant baculovirus budded virus envelopes and liposomes fused with the recombinant virus. Moreover, by using laser-scanning fluorescence microscopy, we observed that GTP analogues specifically bound to Gs alpha subunits on giant liposomes. These results indicate that using the present procedure giant proteoliposomes are prepared with successfully reconstituted G proteins.
Micro-NanoMechatronics and Human Science, 2008. MHS 2008. International Symposium on; 12/2008
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ABSTRACT: Giant unilamellar vesicles (GUVs), or giant liposomes (GLs), are biochemical compartments enclosed with phospholipid bilayers. The size of GUVs is comparable to that of real cells (~10-100 mum). Thus, GUVs are employed to construct artificial cell models, which are reconstituted from biochemical materials to mimic real cellular functions in a simpler manner for investigation of fundamental mechanisms of living systems. However, transmembrane proteins are difficult to reconstitute on GUV bilayers. In order to obtain GUVs containing transmembrane proteins, giant proteoliposomes, here, we introduce the novel method using membrane fusion between GUVs and recombinant baculovirus (Autographa californica nucleopolyhedrovirus (AcNPV)). It is known that the AcNPV budded virus (BV) displays recombinant membrane proteins on their own envelopes. BVs have fusogenic envelope glycoproteins (gp64) sensitive to acidic pH. Combination of these two features may produce giant proteoliposomes. The fusion properties were observed using fluorescence microscopy to comprehend conditions suitable for reconstitution of proteoliposomes. The display of a 7-fold transmembrane receptor protein (G protein-coupled receptor) on GUVs was also demonstrated using fluoroimmuno microscopic observation.
Micro-NanoMechatronics and Human Science, 2007. MHS '07. International Symposium on; 12/2007
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ABSTRACT: Giant unilamellar vesicles (GUVs) are biochemical compartments enclosed with phospholipid bilayers. GUVs are expected to be materials for construction and investigation of artificial cell models. There, fusion phenomena between GUVs and smaller vesicles (referred to as "asymmetric fusion" here) seem to have some potential for utilization of the transporters bringing reagents and membrane proteins to GUVs. In the present work, by using microscopic observation on individual GUVs, we demonstrate asymmetric fusions in the two simple systems: GUVs vs. large unilamellar vesicles (LUVs), and GUVs vs. enveloped virus particles. In the former, the fusion is caused by amphipathic peptides, and we found the condition in which the asymmetric fusion was preferred; the fusion is controllable by having a difference in content ratios of acid lipids between GUVs and LUVs. In the latter, the fusion between GUVs and enveloped virus particles was induced by pH-sensitive fusogenic glycoproteins. The confocal microscopic observation of the asymmetric fusion of GUVs and the particles showed that the particle fused with artificial GUV membranes in naturally preferable conditions: occurring pH and acid lipid contents. The possibilities to preparation giant proteoliposomes by using these systems will be suggested, leading to an application for constructing cell models
Micro-NanoMechatronics and Human Science, 2006 International Symposium on; 12/2006
