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Strategies for successful isolation of a eukaryotic transporter

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Saouros, Savvas, Cecchetti, Cristina, Jones, Alexandra M., Cameron, Alexander and Byrne, Bernadette (2020) Strategies for successful isolation of a eukaryotic transporter. Protein Expression and Purification, 166 . 105522. doi:10.1016/j.pep.2019.105522

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Official URL: http://dx.doi.org/10.1016/j.pep.2019.105522

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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 (OsBOR3 ). 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 OsBOR3 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 OsBOR3 than DDM. Isolation of OsBOR3 in LMNG both in the presence and absence of DTT produced large quantities of protein but contaminated with GFP tagged protein. Isolation of OsBOR3 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 OsBOR3 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. [Abstract copyright: Copyright © 2019 Elsevier Inc. All rights reserved.]

Item Type: Journal Article
Subjects: Q Science > QP Physiology
Divisions: Faculty of Science > Life Sciences (2010- )
SWORD Depositor: Library Publications Router
Library of Congress Subject Headings (LCSH): Membrane proteins, Carrier proteins -- Research, Proteins -- Biological transport, Protein engineering -- Research
Journal or Publication Title: Protein Expression and Purification
Publisher: Academic Press
ISSN: 1046-5928
Official Date: February 2020
Dates:
DateEvent
February 2020Published
23 October 2019Available
20 October 2019Accepted
Date of first compliant deposit: 10 December 2019
Volume: 166
Article Number: 105522
DOI: 10.1016/j.pep.2019.105522
Status: Peer Reviewed
Publication Status: Published
Publisher Statement: ** From PubMed via Jisc Publications Router ** History: received 09-08-2019; revised 18-10-2019; accepted 20-10-2019.
Access rights to Published version: Restricted or Subscription Access
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
UNSPECIFIED[ERC] Horizon 2020 Framework Programmehttp://dx.doi.org/10.13039/100010661
722687H2020 Marie Skłodowska-Curie ActionsUNSPECIFIED
BB/N016467/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268

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