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Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein

Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a “one-size fits all” sys...

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Autores principales: Rottet, Sarah, Iqbal, Shagufta, Beales, Paul A., Lin, Anran, Lee, Jiwon, Rug, Melanie, Scott, Colin, Callaghan, Richard
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284524/
https://www.ncbi.nlm.nih.gov/pubmed/32375237
http://dx.doi.org/10.3390/polym12051049
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author Rottet, Sarah
Iqbal, Shagufta
Beales, Paul A.
Lin, Anran
Lee, Jiwon
Rug, Melanie
Scott, Colin
Callaghan, Richard
author_facet Rottet, Sarah
Iqbal, Shagufta
Beales, Paul A.
Lin, Anran
Lee, Jiwon
Rug, Melanie
Scott, Colin
Callaghan, Richard
author_sort Rottet, Sarah
collection PubMed
description Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a “one-size fits all” system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability; provision of sufficient lateral pressure; and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen; namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds.
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spelling pubmed-72845242020-06-19 Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein Rottet, Sarah Iqbal, Shagufta Beales, Paul A. Lin, Anran Lee, Jiwon Rug, Melanie Scott, Colin Callaghan, Richard Polymers (Basel) Article Investigative systems for purified membrane transporters are almost exclusively reliant on the use of phospholipid vesicles or liposomes. Liposomes provide an environment to support protein function; however, they also have numerous drawbacks and should not be considered as a “one-size fits all” system. The use of artificial vesicles comprising block co-polymers (polymersomes) offers considerable advantages in terms of structural stability; provision of sufficient lateral pressure; and low passive permeability, which is a particular issue for transport assays using hydrophobic compounds. The present investigation demonstrates strategies to reconstitute ATP binding cassette (ABC) transporters into hybrid vesicles combining phospholipids and the block co-polymer poly (butadiene)-poly (ethylene oxide). Two efflux pumps were chosen; namely the Novosphingobium aromaticivorans Atm1 protein and human P-glycoprotein (Pgp). Polymersomes were generated with one of two lipid partners, either purified palmitoyl-oleoyl-phosphatidylcholine, or a mixture of crude E. coli lipid extract and cholesterol. Hybrid polymersomes were characterised for size, structural homogeneity, stability to detergents, and permeability. Two transporters, NaAtm1 and P-gp, were successfully reconstituted into pre-formed and surfactant-destabilised hybrid polymersomes using a detergent adsorption strategy. Reconstitution of both proteins was confirmed by density gradient centrifugation and the hybrid polymersomes supported substrate dependent ATPase activity of both transporters. The hybrid polymersomes also displayed low passive permeability to a fluorescent probe (calcein acetomethoxyl-ester (C-AM)) and offer the potential for quantitative measurements of transport activity for hydrophobic compounds. MDPI 2020-05-03 /pmc/articles/PMC7284524/ /pubmed/32375237 http://dx.doi.org/10.3390/polym12051049 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rottet, Sarah
Iqbal, Shagufta
Beales, Paul A.
Lin, Anran
Lee, Jiwon
Rug, Melanie
Scott, Colin
Callaghan, Richard
Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title_full Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title_fullStr Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title_full_unstemmed Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title_short Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein
title_sort characterisation of hybrid polymersome vesicles containing the efflux pumps naatm1 or p-glycoprotein
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284524/
https://www.ncbi.nlm.nih.gov/pubmed/32375237
http://dx.doi.org/10.3390/polym12051049
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