Rottet Sarah, Iqbal Shagufta, Beales Paul A, Lin Anran, Lee Jiwon, Rug Melanie, Scott Colin, Callaghan Richard
CSIRO Synthetic Biology Future Science Platform, GPO Box 1700, Acton, Canberra 2601, Australia.
Research School of Biology, and the Medical School, Australian National University, Canberra 2601, Australia.
Polymers (Basel). 2020 May 3;12(5):1049. doi: 10.3390/polym12051049.
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 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 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.
用于纯化膜转运蛋白的研究系统几乎完全依赖于使用磷脂囊泡或脂质体。脂质体提供了支持蛋白质功能的环境；然而，它们也有许多缺点，不应被视为“一刀切”的系统。使用包含嵌段共聚物的人工囊泡（聚合物囊泡）在结构稳定性、提供足够的侧向压力以及低被动通透性方面具有相当大的优势，而低被动通透性对于使用疏水化合物的转运测定来说是一个特别的问题。本研究展示了将ATP结合盒（ABC）转运蛋白重构到结合了磷脂和嵌段共聚物聚（丁二烯）-聚（环氧乙烷）的混合囊泡中的策略。选择了两种外排泵；即Atm1蛋白和人P-糖蛋白（Pgp）。用两种脂质配体之一生成聚合物囊泡，要么是纯化的棕榈酰油酰磷脂酰胆碱，要么是脂质提取物和胆固醇的混合物。对混合聚合物囊泡的大小、结构均匀性、对去污剂的稳定性和通透性进行了表征。使用去污剂吸附策略，将两种转运蛋白NaAtm1和P-gp成功重构到预先形成的和经表面活性剂破坏稳定的混合聚合物囊泡中。通过密度梯度离心法确认了两种蛋白质的重构，并且混合聚合物囊泡支持两种转运蛋白的底物依赖性ATP酶活性。混合聚合物囊泡对荧光探针（钙黄绿素乙酰甲氧基酯（C-AM））也表现出低被动通透性，并为定量测量疏水化合物的转运活性提供了潜力。
