Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems

Liguori, Lavinia; Stidder, Barry; Alcaraz, Jean-Pierre; Lenormand, Jean-Luc; Cinquin, Philippe; Martin, Donald K.

doi:10.1080/10826068.2015.1068800

Cited by 6 publications

(6 citation statements)

References 31 publications

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“…All these factors make our protocol an even better strategy than the liposome-fusion proposed by Liguori et al. ( 65 ). These authors set up a method for CF production of VDAC within liposomes that, however, requires much longer preparation times because of the need of preventing aggregation of the integral proteins.…”

Section: Discussionmentioning

confidence: 97%

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Nibali

Rosa

Rauh

et al. 2021

Biophysical Reports

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Voltage-dependent anion-selective channel (VDAC) is one of the main proteins of the outer mitochondrial membrane of all eukaryotes, where it forms aqueous, voltage-sensitive, and ion-selective channels. Its electrophysiological properties have been thoroughly analyzed with the planar lipid bilayer technique. To date, however, available results are based on isolations of VDACs from tissue or from recombinant VDACs produced in bacterial systems. It is well known that the cytosolic overexpression of highly hydrophobic membrane proteins often results in the formation of inclusion bodies containing insoluble aggregates. Purification of properly folded proteins and restoration of their full biological activity requires several procedures that considerably lengthen experimental times. To overcome these restraints, we propose a one-step reaction that combines in vitro cell-free protein expression with nanodisc technology to obtain human VDAC isoforms directly integrated in a native-like lipid bilayer. Reconstitution assays into artificial membranes confirm the reliability of this new methodological approach and provide results comparable to those of VDACs prepared with traditional protein isolation and reconstitution protocols. The use of membrane-mimicking nanodisc systems represents a breakthrough in VDAC electrophysiology and may be adopted to further structural studies.

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Section: Discussionmentioning

confidence: 97%

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Nibali

Rosa

Rauh

et al. 2021

Biophysical Reports

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“…19 We have recently developed and optimized a cell-free expression system capable of overcoming the problem of classical overexpression systems that cannot provide sufficient yields for functional and structural studies of highly complex membrane proteins. 20,21 This E. coli based cell-free expression system is capable of producing milligram amounts of functional membrane proteins of different complexities and from diverse origins. In this paper, we used our optimized cell-free expression system to obtain, in a one-step reaction, recombinant proteoliposomes containing the OprF porin in a native conformation.…”

Section: ■ Introductionmentioning

confidence: 99%

Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers

et al. 2017

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OprF has a central role in Pseudomonas aeruginosa virulence and thus provides a putative target for either vaccines or antibiotic cofactors that could overcome the bacterium's natural resistance to antibiotics. Here we describe a procedure to optimize the production of highly pure and functional OprF porins that are then incorporated into a tethered lipid bilayer. This is a stable biomimetic system that provides the capability to investigate structural aspects and function of OprF using and neutron reflectometry and electrical impedance spectroscopy. The recombinant OprF produced using the optimized cell-free procedure yielded a quantity of between 0.5 to 1.0 mg/mL with a purity ranging from 85 to 91% in the proteoliposomes. The recombinant OprF is capable of binding IFN-γ and is correctly folded in the proteoliposomes. Because OprF proteins form pores the biomimetic system allowed the measurement of OprF conductance using impedance spectroscopy. The neutron reflectometry measurements showed that the OprF protein is incorporated into the lipid bilayer but with parts of the protein in both the regions above and below the lipid bilayer. Those structural aspects are coherent with the current assumed structure of a transmembrane N-terminal domain composed by eight stranded beta-barrels and a globular C-terminal domain located in the periplasm. Currently there are no crystal structures available for OprF. The experimental model system that we describe provides a basis for further fundamental studies of OprF and particularly for the ongoing biotechnological development of OprF as a target for antibacterial drugs.

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“…They can be used as a model membrane system to elucidate the functions and interactions of various cell membranes (Liguori et al. ; Reszczynska et al. ; Wrobel et al.…”

Section: Discussionmentioning

confidence: 99%

“…Liposomes are submicron size vesicles that can be contain natural or synthetic lipids (phospholipids, sterols and/or fatty acids) in their bilayers. They can be used as a model membrane system to elucidate the functions and interactions of various cell membranes (Liguori et al 2015;Reszczynska et al 2015;Wrobel et al 2015), or as a carrier system to deliver biologically active substances into the cells Zhang et al 2015a, 2016Meng et al 2015Zhang et al 2015aZhang et al , 2016 or to incorporate lipids into the plasma membrane (De Silva andSiu 1980, 1981;Hall et al 1991;Wolfe et al 1998;He et al 2001;Christova et al 2004). To effectively achieve these objectives, a high percentage of the intended recipient cells must take up the liposomes.…”

Section: Discussionmentioning

confidence: 99%

Fusion of Boar Sperm with Nanoliposomes Prepared from Synthetic Phospholipids

Kasimanickam

Buhr

2016

Reprod Domestic Animals

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Liposomes are artificial membrane vesicles that can be used to test and model the functions and interactions of various biological membranes, or as a carrier system to deliver biologically active substances into the cells, or to incorporate lipids into the plasma membrane of target cells to modify membrane structure-function relationships. Sperm plasma membrane undergoes lipid modification during maturation in epididymis and during capacitation in the female reproductive tract to facilitate fertilization. Natural variation in the amounts and composition of lipids in the sperm plasma membrane may also contribute to the species-specific sperm sensitivities to handling and storage conditions. Boar sperm are notoriously susceptible to membrane damage and are resistant to compositional alteration by artificial liposomes. This study used flow cytometry to demonstrate stable incorporation of nanoliposomes prepared from a complex mixture of various phospholipids (phosphatidylcholine : phosphatidylethanolamine : sphingomyelin : phosphatidylserine : phosphatidylinositol) with high fusion efficiency. Over 90% of sperm rapidly took up fluorescently labelled liposomes and retained the lipids for at least 60 min, in a significant time- and concentration-dependent manner. This unique fusion efficacy could be used to alter sperm plasma membrane composition and hence membrane-based functional responses.

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Cell-free production of VDAC directly into liposomes for integration with biomimetic membrane systems

Cited by 6 publications

References 31 publications

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Cell-free electrophysiology of human VDACs incorporated into nanodiscs: An improved method

Functional Characterization of Cell-Free Expressed OprF Porin from Pseudomonas aeruginosa Stably Incorporated in Tethered Lipid Bilayers

Fusion of Boar Sperm with Nanoliposomes Prepared from Synthetic Phospholipids

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