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CD81 extracted in SMALP nanodiscs comprises two distinct protein populations within a lipid environment enriched with negatively charged headgroups
- Source :
- Biochimica et Biophysica Acta. Biomembranes
- Publication Year :
- 2020
-
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.<br />Graphical abstract Unlabelled Image<br />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.
- 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
Subjects
Details
- Language :
- English
- ISSN :
- 18792642
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta. Biomembranes
- Accession number :
- edsair.doi.dedup.....76d7f926a89bb5fd1e830f119b5c65a1