Related Experiment Video

Updated: Aug 10, 2026

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa
13:40

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

Published on: February 17, 2014

12.3K

Production of recombinant porin from Y. pseudotuberculosis in a water-soluble form for pseudotuberculosis diagnostics

Vasily Golotin1, Olga Portnyagina1, Natalia Chopenko1

  • 1.

Biological Chemistry
|July 21, 2017
PubMed

Insights

Recombinant OmpF porin from Yersinia pseudotuberculosis was successfully produced and purified. This soluble protein demonstrated ordered structure and efficacy as a diagnostic antigen for pseudotuberculosis detection using ELISA.

Area of Science:

  • Microbiology
  • Structural Biology
  • Immunology

Background:

  • Yersinia pseudotuberculosis is an important human pathogen.
  • Outer membrane proteins like OmpF porin are key virulence factors and potential diagnostic targets.
  • Developing soluble and structurally intact recombinant proteins is crucial for antigen-based diagnostics.

Purpose of the Study:

  • To clone, express, and purify a water-soluble form of OmpF porin from Yersinia pseudotuberculosis.
  • To characterize the structural integrity of the recombinant OmpF porin (rs-OmpF).
  • To evaluate the diagnostic potential of rs-OmpF in detecting pseudotuberculosis.

Main Methods:

  • Gene cloning of Yersinia pseudotuberculosis OmpF porin into pET-40b(+) plasmid.
  • Heterologous expression in E. coli Rosetta (DE3) strain using MX medium and IPTG induction.
  • Optimization of cultivation conditions (0.2 mM IPTG, 14°C overnight) for soluble protein production.
  • Structural analysis of secondary and tertiary structures.
  • Enzyme-Linked Immunosorbent Assay (ELISA) for diagnostic evaluation.

Main Results:

  • Successful cloning and expression of a water-soluble recombinant OmpF porin (rs-OmpF).
  • Rs-OmpF exhibited ordered secondary and tertiary structures, indicating proper folding.
  • ELISA results confirmed the effectiveness of rs-OmpF as a diagnostic antigen for pseudotuberculosis.

Conclusions:

  • The optimized expression system yields structurally sound, water-soluble rs-OmpF.
  • Rs-OmpF is a promising candidate antigen for the development of diagnostic tools for Yersinia pseudotuberculosis infections.
  • This study provides a foundation for further development of serological assays for pseudotuberculosis.
Keywords:
E. coliELISAmembrane proteinsporinsoluble recombinant proteins

More Related Videos

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins09:55

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Published on: July 4, 2016

14.1K
Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth08:36

Quantifying Yersinia pseudotuberculosis Type III Secretion System Activity Following Iron Starvation and Anaerobic Growth

Published on: May 31, 2024

965

Related Concept Videos

Structure of Porins01:21

Structure of Porins

Mitochondria, chloroplasts, and gram-negative bacteria have transmembrane, beta-barrel proteins called porins to mediate the free diffusion of ions and metabolites across the membrane. Mitochondrial porin precursors contain conserved amino acid sequences called beta signals at their C-terminal. Beta signals have a  motif of PoXGXXHyXHy (Po-Polar, X-Any amino acid, G-Glycine, Hy-LargeHydrophobic), which are crucial for precursor recognition to initiate precursor assembly. Beta-barrel precursors...
Production of Pharmaceuticals01:30

Production of Pharmaceuticals

Industrial insulin production uses genetically engineered E. coli expressing a proinsulin gene controlled by a tryptophan promoter and containing a methionine linker for later cleavage. The cells also carry ampicillin resistance for selective growth. Seed cultures are stored at −80 °C and production begins by thawing a small amount to inoculate starter cultures, which are progressively scaled to a 50,000-L bioreactor. In the bioreactor, E. coli grow in nutrient-rich media under sterile, tightly...