Expression of membrane beta-barrel protein in E. coli at low temperatures: Structure of Yersinia pseudotuberculosis OmpF porin inclusion bodies.

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.

Studies on the Structure and Properties of Membrane Phospholipase A Inclusion Bodies Formed at Low Growth Temperatures Using GFP Fusion Strategy.

The effect of cultivation temperatures (37, 26, and 18 °C) on the conformational quality of phospholipase A (PldA) in inclusion bodies (IBs) was studied using green fluorescent protein (GFP) as a folding reporter. GFP was fused to the C-terminus of PldA to form the PldA-GFP chimeric protein. It was found that the maximum level of fluorescence and expression of the chimeric protein is observed in cells grown at 18 °C, while at 37 °C no formation of fluorescently active forms of PldA-GFP occurs.

Chaperone and Immunoglobulin-Binding Activities of Skp Protein from Yersinia pseudotuberculosis.

Here, we determined qualitative and quantitative characteristics of the chaperone and immunoglobulin-binding activities of recombinant Skp protein (rSkp) from Yersinia pseudotuberculosis using the methods of dynamic light scattering and surface plasmon resonance. Commercial human polyclonal IgG and Fc and Fab fragments of human IgG were used as substrate proteins. The activity of rSkp strongly depended on the medium pH.

Inclusion Bodies of Recombinant OmpF Porin from Yersinia pseudotuberculosis: Properties and Structural Characterization.

Mature pore-forming OmpF protein from the outer membrane of Yersinia pseudotuberculosis was expressed in Escherichia coli in the form of inclusion bodies (IBs) under different cultivation conditions. The properties and structural organization of the IBs as well as the structure of the recombinant porin (rOmpF) solubilized from the IBs were investigated using electron microscopy, dynamic light scattering, optical spectroscopy, and specific hydrophobic dyes. The size, shape, and stability of the IBs under denaturing solutions were determined.

Self-Organization of Recombinant Membrane Porin OmpF from Yersinia pseudotuberculosis in Aqueous Environments.

Recombinant porin OmpF (an integral protein of bacterial outer membrane) from Yersinia pseudotuberculosis was synthesized in Escherichia coli cells as inclusion bodies. By combining the methods of anion-exchange and gel filtration chromatographies, recombinant OmpF (rOmpF) was isolated as an individual protein in its denatured state, and its characteristic properties (molecular mass, N-terminal amino acid sequence, and hydrodynamic radius of the protein in 8 M urea solution) were determined. According to the data of gel filtration, dynamic light scattering, optical spectroscopy, and binding of the hydrophobic fluorescent probe 8-anilino-1-naphthalenesulfonic acid, the rOmpF is fully unfolded in 8 M urea and exists in random coil conformation.