1. Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.
- Author
-
Solov'eva, Tamara F., Bakholdina, Svetlana I., Khomenko, Valentina A., Sidorin, Evgeniy V., Kim, Natalya Yu., Novikova, Olga D., Shnyrov, Valery L., Stenkova, Anna M., Eremeev, Vyacheslav I., Bystritskaya, Evgenia P., and Isaeva, Marina P.
- Subjects
- *
YERSINIA pseudotuberculosis , *CELLULAR inclusions , *LOW temperatures , *MEMBRANE proteins , *OPTICAL spectroscopy , *ESCHERICHIA coli - Abstract
The recombinant OmpF porin of Yersinia pseudotuberculosis as a model of transmembrane protein of the β-barrel structural family was used to study low growth temperature effect on the structure of the produced inclusion bodies (IBs). This porin showed a very low expression level in E. coli at a growth temperature below optimal 37 °C. The introduction of a N-terminal hexahistidine tag into the mature porin molecule significantly increased the biosynthesis of the protein at low cultivation temperatures. The recombinant His-tagged porin (rOmpF-His) was expressed in E. coli at 30 and 18 °C as inclusion bodies (IB-30 and IB-18). The properties and structural organization of IBs, as well as the structure of rOmpF-His solubilized from the IBs with urea and SDS, were studied using turbidimetry, electron microscopy, dynamic light scattering, optical spectroscopy, and amyloid-specific dyes. IB-18, in comparison with IB-30, has a higher solubility in denaturants, suggesting a difference between IBs in the conformation of the associated polypeptide chains. The spectroscopic analysis revealed that rOmpF-His IBs have a high content of secondary structure with a tertiary-structure elements, including a native-like conformation, the proportion of which in IB-18 is higher than in IB-30. Solubilization of the porin from IBs is accompanied by a modification of its secondary structure. The studied IBs also contain amyloid-like structures. The results obtained in this study expand our knowledge of the structural organization of IBs formed by proteins of different structural classes and also have a contribution into the new approaches development of producing functionally active recombinant membrane proteins. [Display omitted] • Yersinia pseudotuberculosis OmpF porin forms the inclusion bodies (IBs) when expressed in E. coli. • N-terminal hexahistidine tag is required for efficient expression of the recombinant porin at low cultivation temperatures. • Porin species in native-like conformation are present in IBs and their content increases with decreasing growth temperature. • CD spectroscopy in combination with stepwise IB solubilization provides valid information on the porin secondary structure. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF