Mutant OmpF porins of Yersinia pseudotuberculosis with deletions of external loops: Structure-functional and immunochemical properties

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• 作者：O.V. Sidorova ; ^{siolven@gmail.com" class="auth_mail} ; V.A. Khomenko ; O. Yu Portnyagina ; G.N. Likhatskaya ; T.I. Vakorina ; N. Yu Kim ; D.K. Chistyulin ; T.F. Solov&rsquo ; eva ; O.D. Novikova
• 关键词：Yersinia pseudotuberculosis ; OmpF porin ; Recombinant proteins ; Deletions of the external loops
• 刊名：Biochemical and Biophysical Research Communications
• 出版年：7 March, 2014
• 年：2014
• 卷：445
• 期：2
• 页码：428-432
• 全文大小：594 K

文摘

Recombinant mutant OmpF porins from m>Yersinia pseudotuberculosism> 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 m>Escherichia colim> 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.