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High-level expression and characterization of a purified 142-residue polypeptide of the prion protein
I Mehlhorn1, D Groth, J Stöckel
1Department of Neurology, University of California, San Francisco 94143, USA.
Abstract:
The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.
Insights
Researchers produced high yields of recombinant prion protein (rPrP) in E. coli. This rPrP can adopt multiple conformations, similar to natural prion protein isoforms, aiding structural studies.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Prions are infectious agents composed of misfolded prion proteins (PrPSc).
- Prion protein exists in cellular (PrPC) and scrapie (PrPSc) isoforms, differing in conformation.
- Understanding PrP structure is crucial for prion disease research.
Purpose of the Study:
- To develop a method for high-yield production of recombinant prion protein (rPrP).
- To investigate the conformational properties of the produced rPrP.
- To facilitate structural studies of prion protein isoforms.
Main Methods:
- Cloning and expression of Syrian hamster PrP genes in protease-deficient E. coli.
- Purification of rPrP using size exclusion and reversed-phase chromatography.
- Structural characterization using Edman sequencing, mass spectrometry, circular dichroism, and FTIR spectroscopy.
Main Results:
- Maximal expression achieved for truncated SHaPrP (residues 90-231), yielding ~50 mg/L E. coli culture.
- Purified rPrP under reducing conditions exhibited high beta-sheet content, resembling PrPSc.
- Refolding via oxidation produced a soluble, alpha-helical form similar to PrPC.
Conclusions:
- High-yield production of rPrP is feasible using E. coli expression systems.
- rPrP can adopt distinct conformations (beta-sheet rich or alpha-helical rich), mirroring PrPSc and PrPC.
- This provides a valuable tool for investigating prion protein structure and conformational changes.