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.
|Early online date||28 Jul 2020|
|Publication status||Published - 1 Nov 2020|
Bibliographical note© 2020, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/
Funding: Our work was supported by grants from the Biotechnology & Biosciences Research Council (BBSRC); RMB and MC through BB/N007417/1, IM and AD through BB/S00324X/1 and BB/M006298/1. HA was supported by an Aston University School of Life & Health Sciences Doctoral Studentship, and TK received funding from the German Center for Infection Research (DZIF).
- Membrane protein
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CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups
Ayub, H. (Creator), Clare, M. (Creator), Milic, I. (Creator), Chmel, N. P. (Creator), Böning, H. (Creator), Devitt, A. (Creator), Krey, T. (Creator), Bill, R. (Creator) & Rothnie, A. (Creator), Aston Data Explorer, 19 Dec 2019
DOI: 10.17036/researchdata.aston.ac.uk.00000454, https://www.sciencedirect.com/science/article/pii/S0005273620302625