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Lookup NU author(s): Professor Paul RaceORCiD
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND).
© 2021. Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-β-D-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family.
Author(s): Neves AT, Stenner R, Race PR, Curnow P
Publication type: Article
Publication status: Published
Journal: Protein Expression and Purification
Year: 2022
Volume: 190
Print publication date: 01/02/2022
Online publication date: 01/11/2021
Acceptance date: 31/10/2021
Date deposited: 21/12/2023
ISSN (print): 1046-5928
ISSN (electronic): 1096-0279
Publisher: Academic Press Inc.
URL: https://doi.org/10.1016/j.pep.2021.106011
DOI: 10.1016/j.pep.2021.106011
PubMed id: 34737041
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