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Biochemistry (Moscow)

, Volume 79, Issue 7, pp 694–705 | Cite as

Study of effect of substitution of the penultimate amino acid residue on expression, structure, and functional properties of Yersinia pseudotuberculosis OmpY porin

  • T. F. Solov’evaEmail author
  • N. M. Tischenko
  • V. A. Khomenko
  • O. Y. PortnyaginaEmail author
  • N. Y. Kim
  • G. N. Likhatskaya
  • O. D. Novikova
  • M. P. IsaevaEmail author
Article
  • 58 Downloads

Abstract

The purpose of the study was to compare the expression of two Yersinia pseudotuberculosis proteins, wild-type porin OmpY and the mutant porin OmpY designated as OmpY-Q having the uncharged amino acid residue Gln instead of positively charged Arg at the penultimate position in the same heterologous host. According to the literature, a similar substitution (Lys to Gln) of the penultimate amino acid residue in Neisseria meningitidis porin PorA drastically improved the assembly of the protein in the E. coli outer membrane in vivo. Site-directed mutagenesis was used to replace Arg by Gln (R338Q) in OmpY, and the conditions for optimal expression and maturation of OmpY-Q were selected. It was found that the growth rates of E. coli strains producing OmpY and OmpY-Q and the expression levels of the porins were approximately equal. Comparative analysis of recombinant OmpY and OmpY-Q did not show significant differences in structure, antigenic, and functional properties of the porins, or any noticeable effect of the R338Q substitution in OmpY on its assembly in the E. coli outer membrane in vivo. The probable causes of discrepancies between our results and the previous data on porin PorA are discussed considering the known mechanisms of biogenesis of porins at the periplasmic stage.

Key words

Yersinia pseudotuberculosis outer membrane pore-forming proteins site-directed mutagenesis biogenesis of porins spatial structure computer modeling 

Abbreviations

BLM

bilayer lipid membrane

CM

cytoplasmic membrane

IPTG

isopropyl-β-D-1-thiogalactopyranoside

OM

outer membrane

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Copyright information

© Pleiades Publishing, Ltd. 2014

Authors and Affiliations

  1. 1.Elyakov Pacific Institute of Bioorganic ChemistryFar East Branch of the Russian Academy of SciencesVladivostokRussia

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