1. CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups
- Author
-
Michelle Clare, Alice Rothnie, Heike Böning, Thomas Krey, Roslyn M. Bill, Ivana Milic, Andrew Devitt, Nikola Paul Chmel, and Hoor Ayub
- Subjects
Models, Molecular ,Protein Folding ,viruses ,P. pastoris, Pichia pastoris ,Crystallography, X-Ray ,Biochemistry ,OG, octyl glucoside ,Tetraspanin ,SD, Standard deviation ,EC2, extracellular loop 2 ,PBST, phosphate-buffered saline + Tween 20 (0.05% v/v) ,SDS, sodium dodecyl sulphate ,CD, circular dichroism ,Purification ,DLS, dynamic light scattering ,Infectivity ,chemistry.chemical_classification ,SMALP ,0303 health sciences ,biology ,030302 biochemistry & molecular biology ,HRP, horseradish peroxidase ,SMALP, styrene maleic acid lipid particle ,Recombinant Proteins ,HCV, hepatitis C virus ,Stability ,OPD, o-phenylenediamine dihydrochloride ,ELISA, enzyme linked immune absorbant assay ,SDS-PAGE, SDS polyacrylamide gel electrophoresis ,PBS, phosphate-buffered saline ,Biophysics ,SMA, styrene maleic acid co-polymer ,chemical and pharmacologic phenomena ,SEM, standard error of the mean ,Article ,Tetraspanin 28 ,Pichia pastoris ,03 medical and health sciences ,Humans ,LEL, large extracellular loop ,mAb, monoclonal antibody ,Solubilisation ,030304 developmental biology ,EM, electron microscopy ,SEL, small extracellular loop ,Cell Biology ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,Ni-NTA, nickel nitrilotriacetic acid ,Yeast ,biological factors ,MS, mass spectrometry ,Membrane protein ,chemistry ,Saccharomycetales ,SEC, size exclusion chromatography ,Glycoprotein ,DDM, dodecylmaltoside ,Function (biology) ,CD81 - Abstract
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., Graphical abstract Unlabelled Image, Highlights • CD81 expressed in Pichia pastoris can be solubilised and purified using SMA polymer. • SMALP-encapsulated CD81 retains native folded structure. • Expression and buffer conditions can be optimized to improve protein quality. • The lipid environment surrounding CD81 is enriched with negatively charged lipids.
- Published
- 2020