Strategies for successful isolation of a eukaryotic transporter
File(s)
Accepted version
Author(s)
Saouros, Savvas
Cecchetti, Cristina
Jones, Alex
Cameron, Alexander D
Byrne, Bernadette
Type
Journal Article
Abstract
The isolation of integral membrane proteins for structural analysis remains challenging and this is particularly the case for eukaryotic membrane proteins. Here we describe our efforts to isolate OsBOR3, a boron transporter from Oryza sativa. OsBOR3 was expressed as both full length and a C-terminally truncated form lacking residues 643–672 (OsBOR3Δ1-642). While both express well as C-terminal GFP fusion proteins in Saccharomyces cerevisiae, the full length protein isolates poorly in the detergent dodecyl-β-d-maltoside (DDM). The OsBOR3Δ1-642 isolated in DDM in large quantities but was contaminated with GFP tagged protein, indicated incomplete protease removal of the tag. Addition of the reducing agent dithiothreitol (DTT) had no effect on isolation. Detergent screening indicated that the neopentyl glycol detergents, LMNG, UDMNG and DMNG conferred greater stability on the OsBOR3Δ1-642 than DDM. Isolation of OsBOR3Δ1-642 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3Δ1-642 in DMNG + DTT resulted in protein sample that does not contain any detectable GFP but elutes at a higher retention volume than that seen for protein isolated in either DDM or LMNG. Mass spectrometry confirmed that the LMNG and DMNG purified protein is OsBOR3Δ1-642 indicating that the DMNG isolated protein is monomer compared to the dimer isolated using LMNG. This was further supported by single particle electron microscopic analysis revealing that the DMNG protein particles are roughly half the size of the LMNG protein particles.
Date Issued
2020-02-01
Date Acceptance
2019-10-20
Citation
Protein Expression and Purification, 2020, 166, pp.1-8
ISSN
1046-5928
Publisher
Elsevier
Start Page
1
End Page
8
Journal / Book Title
Protein Expression and Purification
Volume
166
Copyright Statement
© 2019 Elsevier Ltd. All rights reserved. This manuscript is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International Licence http://creativecommons.org/licenses/by-nc-nd/4.0/
Sponsor
Biotechnology and Biological Sciences Research Council (BBSRC)
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000500362800013&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Grant Number
BB/K017292/1
Subjects
Science & Technology
Life Sciences & Biomedicine
Biochemical Research Methods
Biochemistry & Molecular Biology
Biotechnology & Applied Microbiology
BOR protein
Transporter
Truncation
Purification
Detergent
Neopentyl glycol
Stability
Oligomeric state
MEMBRANE-PROTEINS
BORON TRANSPORTER
CRYSTALLIZATION
DETERGENTS
STABILIZATION
PURIFICATION
AMPHIPHILES
OVEREXPRESSION
SOLUBILIZATION
OPTIMIZATION
Publication Status
Published
Article Number
ARTN 105522
Date Publish Online
2019-10-23