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Production, purification and oxidative folding of the mouse recombinant prion protein

A Pavlícek1, L Bednárová, K Holada

  • 1Institute of Microbiology and Immunology, 1st Faculty of Medicine, Charles University, 128 00 Prague, Czechia.

Folia Microbiologica
|December 8, 2007
PubMed

Insights

Researchers developed a method to produce and purify mouse recombinant prion protein (mrPrP) in E. coli. The purified protein

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Prion protein (PrP) misfolding is linked to neurodegenerative diseases.
  • Efficient production of recombinant PrP is crucial for studying its structure and function.
  • Bacterial expression systems offer a scalable platform for protein production.

Purpose of the Study:

  • To establish a method for overexpressing and purifying full-length mouse recombinant prion protein (mrPrP 23-231) in E. coli.
  • To characterize the folding properties of the purified mrPrP.

Main Methods:

  • Overexpression of his-mrPrP fusion protein in E. coli cytoplasm.
  • Purification using affinity chromatography.
  • Solubilization and refolding of purified mrPrP.
  • Analysis of protein conformation using circular dichroism, proteinase K digestion, and sucrose gradient centrifugation.

Main Results:

  • Achieved a typical yield of 8-10 mg of his-mrPrP per liter of bacterial culture.
  • Obtained purified protein with >95% purity.
  • Demonstrated the ability to convert purified mrPrP to a soluble form.
  • Characterized the folding into alpha-helical and beta-sheet conformations and their distinct properties.

Conclusions:

  • A robust method for producing highly pure mouse recombinant prion protein in E. coli was developed.
  • The purified mrPrP can be refolded into different conformations.
  • The study provides a foundation for further investigations into prion protein structure-function relationships and disease mechanisms.

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