Department of Microbiology, Biomedicine Discovery Institute, Monash University, Melbourne, Victoria 3800, Australia.
Department of Biochemistry and Molecular Biology, Monash University, Melbourne, Victoria 3800, Australia.
Biosci Rep. 2024 Sep 25;44(9). doi: 10.1042/BSR20240692.
Motility by means of flagella plays an important role in the persistent colonization of Helicobacter pylori in the human stomach. The H. pylori flagellar motor has a complex structure that includes a periplasmic scaffold, the components of which are still being identified. Here, we report the isolation and characterization of the soluble forms of two putative essential H. pylori motor scaffold components, proteins PflA and PflB. We developed an on-column refolding procedure, overcoming the challenge of inclusion body formation in Escherichia coli. We employed mild detergent sarkosyl to enhance protein recovery and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO)-containing buffers to achieve optimal solubility and monodispersity. In addition, we showed that PflA lacking the β-rich N-terminal domain is expressed in a soluble form, and behaves as a monodisperse monomer in solution. The methods for producing the soluble, folded forms of H. pylori PflA and PflB established in this work will facilitate future biophysical and structural studies aimed at deciphering their location and their function within the flagellar motor.
鞭毛的运动能力在幽门螺杆菌（Helicobacter pylori）在人类胃部的持续定植中起着重要作用。幽门螺杆菌鞭毛马达具有复杂的结构，包括周质支架，其组成部分仍在鉴定中。在这里，我们报告了两种假定的必需幽门螺杆菌马达支架成分，蛋白 PflA 和 PflB 的可溶性形式的分离和表征。我们开发了一种在柱上复性的程序，克服了在大肠杆菌中形成包涵体的挑战。我们使用温和的去污剂 Sarkosyl 来提高蛋白质回收率，并使用含 n-十二烷基-N,N-二甲基氧化胺（LDAO）的缓冲液来实现最佳的可溶性和单分散性。此外，我们还表明，缺乏富含 β 的 N 端结构域的 PflA 以可溶形式表达，并在溶液中表现为单分散的单体。本工作中建立的生产幽门螺杆菌 PflA 和 PflB 可溶性、折叠形式的方法将有助于未来的生物物理和结构研究，以阐明它们在鞭毛马达中的位置和功能。
