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...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-05-01
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| Series: | Polymers |
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| Online Access: | https://www.mdpi.com/2073-4360/12/5/1049 |
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| author | Sarah Rottet Shagufta Iqbal Paul A. Beales Anran Lin Jiwon Lee Melanie Rug Colin Scott Richard Callaghan |
| author_facet | Sarah Rottet Shagufta Iqbal Paul A. Beales Anran Lin Jiwon Lee Melanie Rug Colin Scott Richard Callaghan |
| author_sort | Sarah Rottet |
| collection | DOAJ |
| 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 <i>Novosphingobium aromaticivorans</i> 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 <i>E. coli</i> 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. |
| first_indexed | 2024-03-10T20:03:43Z |
| format | Article |
| id | doaj.art-96683e2a777245bfb4398c63e6591c8c |
| institution | Directory Open Access Journal |
| issn | 2073-4360 |
| language | English |
| last_indexed | 2024-03-10T20:03:43Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Polymers |
| spelling | doaj.art-96683e2a777245bfb4398c63e6591c8c2023-11-19T23:24:55ZengMDPI AGPolymers2073-43602020-05-01125104910.3390/polym12051049Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-GlycoproteinSarah Rottet0Shagufta Iqbal1Paul A. Beales2Anran Lin3Jiwon Lee4Melanie Rug5Colin Scott6Richard Callaghan7CSIRO Synthetic Biology Future Science Platform, GPO Box 1700, Acton, Canberra 2601, AustraliaResearch School of Biology, and the Medical School, Australian National University, Canberra 2601, AustraliaSchool of Chemistry Bragg Centre for Materials Research and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UKResearch School of Biology, and the Medical School, Australian National University, Canberra 2601, AustraliaCentre for Advanced Microscopy, Australian National University, Canberra 2601, AustraliaCentre for Advanced Microscopy, Australian National University, Canberra 2601, AustraliaCSIRO Synthetic Biology Future Science Platform, GPO Box 1700, Acton, Canberra 2601, AustraliaResearch School of Biology, and the Medical School, Australian National University, Canberra 2601, AustraliaInvestigative 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 <i>Novosphingobium aromaticivorans</i> 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 <i>E. coli</i> 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.https://www.mdpi.com/2073-4360/12/5/1049ATP binding cassettereconstitutionmembrane proteinpolymersomesliposomespoly (butadiene)-poly (ethylene oxide) |
| spellingShingle | Sarah Rottet Shagufta Iqbal Paul A. Beales Anran Lin Jiwon Lee Melanie Rug Colin Scott Richard Callaghan Characterisation of Hybrid Polymersome Vesicles Containing the Efflux Pumps NaAtm1 or P-Glycoprotein Polymers ATP binding cassette reconstitution membrane protein polymersomes liposomes poly (butadiene)-poly (ethylene oxide) |
| 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 | ATP binding cassette reconstitution membrane protein polymersomes liposomes poly (butadiene)-poly (ethylene oxide) |
| url | https://www.mdpi.com/2073-4360/12/5/1049 |
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