Expression, purification and preliminary characterisation of the choline transporter LicB from opportunistic bacterial pathogens.
Protein Expr Purif
; 190: 106011, 2022 02.
Article
in En
| MEDLINE
| ID: mdl-34737041
ABSTRACT
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-ß-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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Membrane Transport Proteins
/
Bacterial Proteins
/
Gene Expression
/
Haemophilus influenzae
Language:
En
Journal:
Protein Expr Purif
Journal subject:
BIOLOGIA MOLECULAR
Year:
2022
Document type:
Article
Affiliation country:
United kingdom