CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups.
Membrane protein
Purification
SMALP
Solubilisation
Stability
Tetraspanin
Journal
Biochimica et biophysica acta. Biomembranes
ISSN: 1879-2642
Titre abrégé: Biochim Biophys Acta Biomembr
Pays: Netherlands
ID NLM: 101731713
Informations de publication
Date de publication:
01 11 2020
01 11 2020
Historique:
received:
19
12
2019
revised:
05
06
2020
accepted:
07
07
2020
pubmed:
1
8
2020
medline:
30
12
2020
entrez:
1
8
2020
Statut:
ppublish
Résumé
Tetraspanins exert a wide range of cellular functions of broad medical importance. Despite this, their biophysical characteristics are incompletely understood. Only two high-resolution structures of full-length tetraspanins have been solved. One is that of human CD81, which is involved in the infectivity of human pathogens including influenza, HIV, the malarial Plasmodium parasite and hepatitis C virus (HCV). The CD81 crystal structure identifies a cholesterol-binding pocket, which has been suggested to be important in the regulation of tetraspanin function. Here we investigate the use of styrene-maleic anhydride co-polymers (SMA) for the solubilisation and purification of CD81 within a lipid environment. When CD81 was expressed in the yeast Pichia pastoris, it could be solubilised and purified using SMA2000. This SMALP-encapsulated CD81 retained its native folded structure, as determined by the binding of two conformation-sensitive anti-CD81 antibodies. Analysis by size exclusion chromatography revealed two distinct populations of CD81, only one of which bound the HCV glycoprotein, E2. Optimization of expression and buffer conditions increased the proportion of E2-binding competent CD81 protein. Mass spectrometry analysis indicated that the lipid environment surrounding CD81 is enriched with negatively charged lipids. These results establish a platform to study the influence of protein-lipid interactions in tetraspanin biology.
Identifiants
pubmed: 32735789
pii: S0005-2736(20)30262-5
doi: 10.1016/j.bbamem.2020.183419
pmc: PMC7456796
pii:
doi:
Substances chimiques
CD81 protein, human
0
Recombinant Proteins
0
Tetraspanin 28
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
183419Subventions
Organisme : Biotechnology & Biosciences Research Council
ID : BB/N007417/1
Pays : International
Organisme : Biotechnology & Biosciences Research Council
ID : BB/S00324X/1
Pays : International
Organisme : Biotechnology & Biosciences Research Council
ID : BB/M006298/1
Pays : International
Informations de copyright
Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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