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Biochemistry (Moscow)

, Volume 84, Issue 6, pp 672–685 | Cite as

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

  • V. A. Khomenko
  • E. V. Sidorin
  • S. I. Bakholdina
  • G. A. Naberezhnykh
  • N. Yu. Kim
  • A. M. Stenkova
  • N. Yu. Chernysheva
  • M. P. Isaeva
  • T. F. Solov’evaEmail author
Article
  • 3 Downloads

Abstract

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. It was found that the IBs were readily soluble in SDS and more resistant to urea. Dissolution of the IBs in both denaturing agents led to formation of a heterogeneous in size population of oligomeric particles. The IBs contained an intermediate form of the rOmpF with native-like secondary structure and elements of tertiary structure, which was able to penetrate a lipid bilayer and adopt a functionally active conformation. There were no significant differences in the properties and structure between the examined IBs formed at different concentrations of the inducer (IPTG). However, the content of amyloids in the IBs increased with increasing concentration of the inducer. These results contribute to the development of new approaches for the production of active proteins from IBs, as well as biologically and functionally active IBs.

Keywords

Yersinia pseudotuberculosis inclusion bodies recombinant OmpF porin dynamic light scattering electron microscopy CD spectroscopy fluorescence spectroscopy 

Abbreviations

ANS

8-anilinonaphthalene-1-sulfonic acid

CF

carboxyfluorescein

DLS

dynamic light scattering

IBs

inclusion bodies

IPTG

isopropyl-β-D-1-thiogalactopyranoside

PDI

polydispersity index

PMSF

phenylmethylsulfonyl fluoride

RH

hydrodynamic radius

rOmpF

recombinant OmpF protein

SB3-12

zwitterionic detergent (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate)

SMR

specific marker release

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Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  • V. A. Khomenko
    • 1
  • E. V. Sidorin
    • 1
  • S. I. Bakholdina
    • 1
  • G. A. Naberezhnykh
    • 1
  • N. Yu. Kim
    • 1
  • A. M. Stenkova
    • 1
  • N. Yu. Chernysheva
    • 1
  • M. P. Isaeva
    • 1
  • T. F. Solov’eva
    • 1
    Email author
  1. 1.Elyakov Pacific Institute of Bioorganic ChemistryFar Eastern Branch of the Russian Academy of SciencesVladivostokRussia

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