Construction of in vitro reconstitution system for inverse autotransporter Intimin
10.3760/cma.j.cn112309-20230301-00048
- VernacularTitle:反式自主转运蛋白紧密黏附素Intimin离体重组系统的建立
- Author:
Liangwan WEI
1
;
Zhe WANG
;
Xingyuan WANG
;
Yindi CHU
;
Enguo FAN
Author Information
1. 中国医学科学院基础医学研究所北京协和医学院基础学院病原学系，医学分子生物学国家重点实验室，北京 100005
- Keywords:
Inverse autotransporter;
BAM complex;
Membrane translocation
- From:
Chinese Journal of Microbiology and Immunology
2023;43(3):191-195
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To construct an in vitro reconstitution system for inverse autotransporters in order to further investigate their translocation mechanism. Methods:Intimin from Escherichi coli was used as a model substrate. Spheroplasts were prepared from Escherichi coli strains overexpressing Intimin to induce the expression of Intimin. Recombinant β-barrel assembly machinery (BAM) complex was obtained and purified, and then proteoliposomes containing BAM were prepared. Following the digestion with proteinase K, the translocation was detected by SDS-PAGE. Results:Spheroplasts were induced to express Intimin, and then BAM-containing proteoliposomes were added to the system. Compared with control and liposomes groups, the experimental group showed that Intimin was resistant to proteinase K treatment, indicating that Intimin was successfully translocated.Conclusions:The translocation of Intimin required the participation of BAM complex. An in vitro reconstitution system for inverse autotransporters was constructed in this study, providing a method to study the translocation mechanism of inverse autotransporters.
