title: Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: structure-functional and immunochemical properties.
abstract: Recombinant mutant OmpF porins from Yersinia pseudotuberculosis outer membrane were obtained using site-directed mutagenesis. Here we used four OmpF mutants where single extracellular loops L1, L4, L6, and L8 were deleted one at a time. The proteins were expressed in Escherichia coli at levels comparable to full-sized recombinant OmpF porin and isolated from the inclusion bodies. Purified trimers of the mutant porins were obtained after dialysis and consequent ion-exchange chromatography. Changes in molecular and spatial structure of the mutants obtained were studied using SDS-PAGE and optical spectroscopy (circular dichroism and intrinsic protein fluorescence). Secondary and tertiary structure of the mutant proteins was found to have some features in comparison with that of the full-sized recombinant OmpF. As shown by bilayer lipid membrane technique, the pore-forming activity of purified mutant porins was identical to OmpF porin isolated from the bacterial outer membrane. Lacking of the external loops mentioned above influenced significantly upon the antigenic structure of the porin as demonstrated using ELISA.
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia. Electronic address: siolven@gmail.com.
authors: O V
authors: OV
authors: Sidorova
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: V A
authors: VA
authors: Khomenko
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: O Yu
authors: OY
authors: Portnyagina
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: G N
authors: GN
authors: Likhatskaya
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: T I
authors: TI
authors: Vakorina
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: N Yu
authors: NY
authors: Kim
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: D K
authors: DK
authors: Chistyulin
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: T F
authors: TF
authors: Solov'eva
authors: G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch, Russian Academy of Sciences, Prospect 100 let Vladivostoku 159, Vladivostok 690022, Russia.
authors: O D
authors: OD
authors: Novikova
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D003001:Cloning, Molecular; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018272:Porins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D015011:Yersinia pseudotuberculosis / Q000737:chemistry / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D015012:Yersinia pseudotuberculosis Infections / Q000276:immunology / Q000382:microbiology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; C109237:OmpF protein; D018272:Porins; D011994:Recombinant Proteins
keywords: Deletions of the external loops; OmpF porin; Recombinant proteins; Yersinia pseudotuberculosis