Neves Angela Tf, Stenner Richard, Race Paul R, Curnow Paul
School of Biochemistry, University of Bristol, UK.
School of Biochemistry, University of Bristol, UK.
Protein Expr Purif. 2022 Feb;190:106011. doi: 10.1016/j.pep.2021.106011. Epub 2021 Nov 1.
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.
许多感染上呼吸道的机会致病菌会在其细胞表面修饰磷酰胆碱，以支持细菌的定植和生长。这些表面修饰需要从宿主环境中主动摄取胆碱，这一过程被认为是由一类作为胆碱通透酶的专用整合膜蛋白介导的。在此，我们展示了这些胆碱转运蛋白原型——来自流感嗜血杆菌的LicB的表达与纯化。我们表明，LicB可以在大肠杆菌中重组表达，并使用去污剂正十二烷基-β-D-麦芽糖苷以稳定的折叠状态纯化至同质。用荧光配体丹磺酰胆碱进行的平衡结合研究表明，LicB对胆碱具有选择性，对乙酰胆碱的亲和力降低，对包括甘氨酸、肉碱和甜菜碱在内的其他小分子没有明显活性。我们还在LicB家族中鉴定出一个保守的序列基序，并表明该基序内的突变会损害蛋白质结构和功能。我们的结果与之前关于LicB是一种特异性高亲和力胆碱转运蛋白的观察结果一致，并为进一步研究该通透酶家族提供了一个实验平台。
