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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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WRAP-Strategies-successful-isolation-eukaryotic-transporter-jones-2019.pdf - Accepted Version - Requires a PDF viewer. Available under License Creative Commons Attribution Non-commercial No Derivatives 4.0. Download (973Kb) | Preview |
Official URL: http://dx.doi.org/10.1016/j.pep.2019.105522
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 | ||||||||||||
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| Subjects: | Q Science > QP Physiology | ||||||||||||
| Divisions: | Faculty of Science, Engineering and Medicine > 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: |
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| Volume: | 166 | ||||||||||||
| Article Number: | 105522 | ||||||||||||
| DOI: | 10.1016/j.pep.2019.105522 | ||||||||||||
| Status: | Peer Reviewed | ||||||||||||
| Publication Status: | Published | ||||||||||||
| Reuse Statement (publisher, data, author rights): | ** 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: |
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