Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer.
Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer.
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使用二异丁基马来酸（DIBMA）共聚物对功能性人类5-羟色胺转运蛋白（SERT）的无洗涤剂纯化和重构。
DOI：
10.1016/j.bbamem.2021.183602
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发表时间：
2021-07-01
期刊：
影响因子：
--
通讯作者：
Booth PJ
中科院分区：
文献类型：
--
作者：
Dilworth MV;Findlay HE;Booth PJ
尽管这些蛋白质在细胞基础中的重要性及其在药物靶点中的优势地位，但在脂质双层环境中对人类膜蛋白的结构和功能的分析严重缺乏。一个潜在的原因是，详细的研究通常需要在随后在膜模拟物中重构之前从其宿主膜中对蛋白质进行潜在的不稳定去污剂纯化;由于过度表达适量蛋白质的固有困难，这种情况对于人膜蛋白质来说是恶化的。我们提出了有前途的苯乙烯马来酸聚合物（SMA）提取方法，介绍了一种无洗涤剂的方法，直接从酵母细胞中获得稳定的，功能性的人类膜转运蛋白的双层纳米盘。我们纯化人血清素转运蛋白（hSERT）后，在毕赤酵母使用二异丁烯马来酸（DIBMA）作为一个上级的方法，传统的洗涤剂或更确定的苯乙烯马来酸聚合物过表达。hSERT在神经递质调节中起关键作用，负责神经递质5-羟色胺（5-HT或5-羟色胺）的转运。它是神经递质钠同向转运体（NSS）家族的代表，其重要性因其功能障碍而导致的众多疾病而凸显。我们通过体外转运试验深入了解hSERT活性，发现DIBMA提取物比常规洗涤剂方法提高了hSERT的热稳定性和活性。非芳香族两亲性聚合物DIBMA可成功地用于纯化人膜蛋白。来自酵母膜的hSERT的DIBMA增溶和所得纳米盘热稳定性与SMA相当。DIBMA和SMA包封的hSERT脂质纳米盘表现出比hSERT DDMCHS胶束更高的结合活性。用hSERT-DIBMALP重建的蛋白脂质体比类似的DDMCHS重建具有更高的转运活性。
Structure and function analysis of human membrane proteins in lipid bilayer environments is acutely lacking despite the fundame1ntal cellular importance of these proteins and their dominance of drug targets. An underlying reason is that detailed study usually requires a potentially destabilising detergent purification of the proteins from their host membranes prior to subsequent reconstitution in a membrane mimic; a situation that is exacerbated for human membrane proteins due to the inherent difficulties in overexpressing suitable quantities of the proteins. We advance the promising styrene maleic acid polymer (SMA) extraction approach to introduce a detergent-free method of obtaining stable, functional human membrane transporters in bilayer nanodiscs directly from yeast cells. We purify the human serotonin transporter (hSERT) following overexpression in Pichia pastoris using diisobutylene maleic acid (DIBMA) as a superior method to traditional detergents or the more established styrene maleic acid polymer. hSERT plays a pivotal role in neurotransmitter regulation being responsible for the transport of the neurotransmitter 5-hydroxytryptamine (5-HT or serotonin). It is representative of the neurotransmitter sodium symporter (NSS) family, whose importance is underscored by the numerous diseases attributed to their malfunction. We gain insight into hSERT activity through an in vitro transport assay and find that DIBMA extraction improves the thermostability and activity of hSERT over the conventional detergent method. The non-aromatic amphipathic polymer DIBMA can be successfully employed to purify human membrane proteins. DIBMA solubilisation of hSERT from yeast membranes and resultant nanodisc thermostability is comparable to SMA. DIBMA and SMA encapsulated hSERT lipid-nanodiscs exhibit higher binding activity than hSERT DDMCHS micelles. Proteoliposomes reconstituted with hSERT-DIBMALPs possess higher transport activity than comparable DDMCHS reconstitutions.
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影响因子：
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作者：
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