Yersinia pseudotuberculosis mutant OmpF porins with deletions of the external loops: genetic constructions design, expression, isolation and refolding

• Published in: Russian journal of bioorganic chemistry 2012-03, Vol.38 (2), p.133-141
• Main Authors: Sidorova, O. V., Isaeva, M. P., Khomenko, V. A., Portnyagina, O. Yu, Likhatskaya, G. N., Kim, N. Yu, Novikova, O. D., Chistyulin, D. K., Solov’eva, T. F.
• Format: Article
• Language: eng ; rus
• Subjects: Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Dialysis; Enzyme-linked immunosorbent assay; Escherichia coli; Gene deletion; Immunoblotting; Inclusion bodies; Ion-exchange chromatography; Life Sciences; Lipid membranes; OmpF protein; Organic Chemistry; Outer membranes; Plasmids; Porins; Protein structure; Pseudotuberculosis; Site-directed mutagenesis; Tertiary structure; Yersinia pseudotuberculosis
• ISSN: 1068-1620; 1608-330X
• DOI: 10.1134/S1068162012010189

Yersinia pseudotuberculosis outer membrane (OM) recombinant mutant OmpF porins with deletions of the external loops L1, L6 and L8 were obtained using site-directed mutagenesis of the recombinant plasmid including ompF gene. Heterologeous expression of the mutant proteins was carried out in strain Rosetta of Escherichia coli (Novagen, USA), porins with the deletions were isolated from the inclusion bodies. Oligomers of mutant porins were obtained as result of dialysis and ion-exchange chromatography. Spatial structure of the mutant proteins was found to have special features in comparison with that of the full-structured OmpF porin on the level of both secondary and tertiary structure. As shown using bilayer lipid membrane (BLM) technique the absence of the loops L1, L6 and L8 didn’t affect the conductivity level of Y. pseudotuberculosis porin channel. The absence of the loops mentioned above has a significant influence on the antigenic structure of the mutant porins as demonstrated using immunoblotting technique and ELISA.