Cell‐Free Preparation of Functional and Triggerable Giant Proteoliposomes

Liu, Yanjun; Hansen, G.; Venancio‐Marques, Anna; Baigl, Damien

doi:10.1002/cbic.201300501

Cited by 31 publications

(34 citation statements)

References 55 publications

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“…GUVs containing proteins, was a very challenging task. Several protocols have been proposed 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , mostly based on the ones established for protein-free GUVs. All these approaches have been successfully used with specific proteins.…”

Section: Introductionmentioning

confidence: 99%

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

et al. 2015

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Giant unilamellar vesicles (GUVs), composed of a phospholipid bilayer, are often used as a model system for cell membranes. However the study of proteo-membrane interactions in this system is limited as the incorporation of integral and lipid-anchored proteins into GUVs remains challenging. Here, we present a simple generic method to incorporate proteins into GUVs. The basic principle is to break proteo-liposomes by an osmotic shock. They subsequently reseal into larger vesicles which, if necessary, can endure the same to obtain even bigger proteo-GUVs. This process does not require specific lipids or reagents, works under physiological conditions with high concentrations of protein, the proteins remains functional after incorporation and the resulting proteo-GUVs can be micromanipulated. Moreover, our protocol is valid for a wide range of protein substrates. We have successfully reconstituted three structurally different proteins, two trans-membrane proteins, TolC and the neuronal t-SNARE, and one lipid-anchored peripheral protein, GABARAP-Like 1 (GL1). In each case, we verified that the protein remains active after incorporation and in their correctly folded state. We also measured their mobility by performing diffusion measurements via fluorescence recovery after photobleaching (FRAP) experiments on micromanipulated single GUVs. The diffusion coefficients are in agreement with previous data.

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Section: Introductionmentioning

confidence: 99%

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

et al. 2015

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“…We expressed Cx43-eGFP (Cx43 fused with enhanced green fluorescent protein (eGFP)) using an in vitro protein synthesis system outside the GVs. We then measured the fluorescence intensity of Cx43-eGFP on these vesicles 34 . As a result, the fluorescent Cx43-eGFP was observed for each of the components of the GV membranes after incubation at 37°C for two hours (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The GVs that contained Cx43-eGFP were observed using CLSM with an oilimmersion lens (×60) and the fluorescence at 505-530 nm excitation was measured using a diode laser (473 nm) (FV-1200, Oylmpus). The fluorescence intensity on the surface of the GVs was measured using a radial profile of ImageJ software 34 and was obtained from the observed peak value. The leakage of rhodamine 101 (10 µg ml -1 ) into the GVs was observed using CLSM with an oil-immersion lens (×60) and the fluorescence at 560-615 nm excitation was measured using a diode laser (559 nm).…”

Section: Methodsmentioning

confidence: 99%

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

et al. 2016

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Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer. The asymmetric giant vesicles showed a lipid flip-flop behaviour in the membrane, superficially similar to the lipid flip-flop activity observed in apoptotic cells. In vitro synthesis of membrane proteins into the asymmetric giant vesicles revealed that the lipid asymmetry in bilayer membranes improves the reconstitution ratio of membrane proteins. Our asymmetric giant vesicles will be useful in elucidating lipid-lipid and lipid-membrane protein interactions involved in the regulation of cellular functions.

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“…Au-delà de l'encapsulation, la construction d'une membrane active est un défi. Des (Figure 3) [55][56][57]. La réalisation de fonctions plus complexes comme la division ou la réplication de l'ADN semble encore lointaine.…”

Section: Les Systèmes Acellulaires D'expression Comme Plateforme Expéunclassified

Construction de cellules synthétiques

Noireaux

2015

Med Sci (Paris)

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Cell‐Free Preparation of Functional and Triggerable Giant Proteoliposomes

Cited by 31 publications

References 55 publications

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

Formation of Giant Unilamellar Proteo-Liposomes by Osmotic Shock

Cell-sized asymmetric lipid vesicles facilitate the investigation of asymmetric membranes

Construction de cellules synthétiques

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