Detergent-free purification and reconstitution of functional human serotonin transporter (SERT) using diisobutylene maleic acid (DIBMA) copolymer
| Autor: | Paula J. Booth, Marvin V. Dilworth, Heather E. Findlay |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Serotonin
Transport assay Maleic acid Polymers Protein thermostability DIBMA Biophysics Alkenes Biochemistry Article Pichia pastoris 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Humans SMA Neurotransmitter sodium symporter Lipid bilayer Serotonin transporter 030304 developmental biology Serotonin Plasma Membrane Transport Proteins 0303 health sciences biology Chemistry Protein Stability SERT Maleates Temperature Cell Biology biology.organism_classification Yeast Recombinant Proteins 3. Good health Membrane Membrane protein biology.protein 030217 neurology & neurosurgery |
| Zdroj: | Biochimica et Biophysica Acta. Biomembranes |
| ISSN: | 1879-2642 |
| Popis: | 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. Graphical abstract Unlabelled Image Highlights • 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. |
| Databáze: | OpenAIRE |
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