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Article

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

1
CSIRO Synthetic Biology Future Science Platform, GPO Box 1700, Acton, Canberra 2601, Australia
2
Research School of Biology, and the Medical School, Australian National University, Canberra 2601, Australia
3
School of Chemistry Bragg Centre for Materials Research and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
4
Centre for Advanced Microscopy, Australian National University, Canberra 2601, Australia
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Polymers 2020, 12(5), 1049; https://doi.org/10.3390/polym12051049
Received: 8 April 2020 / Revised: 24 April 2020 / Accepted: 25 April 2020 / Published: 3 May 2020
(This article belongs to the Special Issue Hybrid Polymer/Lipid Membranes and Thin Films)
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. View Full-Text
Keywords: ATP binding cassette; reconstitution; membrane protein; polymersomes; liposomes; poly (butadiene)-poly (ethylene oxide); block co-polymer ATP binding cassette; reconstitution; membrane protein; polymersomes; liposomes; poly (butadiene)-poly (ethylene oxide); block co-polymer
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MDPI and ACS Style

Rottet, S.; Iqbal, S.; Beales, P.A.; Lin, A.; Lee, J.; Rug, M.; Scott, C.; Callaghan, R. Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein. Polymers 2020, 12, 1049. https://doi.org/10.3390/polym12051049

AMA Style

Rottet S, Iqbal S, Beales PA, Lin A, Lee J, Rug M, Scott C, Callaghan R. Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein. Polymers. 2020; 12(5):1049. https://doi.org/10.3390/polym12051049

Chicago/Turabian Style

Rottet, Sarah, Shagufta Iqbal, Paul A. Beales, Anran Lin, Jiwon Lee, Melanie Rug, Colin Scott, and Richard Callaghan. 2020. "Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein" Polymers 12, no. 5: 1049. https://doi.org/10.3390/polym12051049

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