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Expression and structural characterization of the recombinant human doppel protein
1Institute of Pathology and Mass Spectrometry Core Facility, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106, USA.
Abstract:
The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like molecule encoded by a novel gene locus, prnd, located on the same chromosome as the PrP gene. To study the structural features of Dpl, we have expressed recombinant human Dpl corresponding to the putative mature protein domain (residues 24-152) in Escherichia coli. The primary structure of the recombinant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edman sequencing, matrix-assisted laser desorption ionization mass spectrometry, and electrospray ionization mass spectrometry. Dpl 24-152 was shown to contain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary structure of Dpl was analyzed using far-UV circular dichroism spectroscopy. Dpl 24-152 was found to be an alpha-helical protein having a high helical content (40%). Dpl 24-152 exhibited characteristics of a thermodynamically stable protein that undergoes reversible and cooperative thermal denaturation. In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar to those of recombinant PrP. This study provides knowledge about the molecular features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.
Insights
The doppel protein (Dpl) is a prion protein-like molecule. Researchers characterized recombinant human Dpl, finding it stable, alpha-helical, and similar to PrP, aiding neurodegenerative disease research.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- The doppel protein (Dpl) is a novel prion protein (PrP)-like molecule.
- Dpl is encoded by the prnd gene, located near the PrP gene.
Purpose of the Study:
- To investigate the structural and biochemical properties of recombinant human Dpl.
- To compare the characteristics of Dpl with those of PrP.
Main Methods:
- Expression of recombinant human Dpl (residues 24-152) in E. coli.
- Characterization using gel electrophoresis, Edman sequencing, mass spectrometry (MALDI-TOF, ESI).
- Secondary structure analysis via far-UV circular dichroism spectroscopy; thermal denaturation studies.
Main Results:
- Recombinant Dpl 24-152 contains two disulfide bonds (Cys94-Cys145 and Cys108-Cys140).
- Dpl 24-152 is an alpha-helical protein (40% helical content) and is thermodynamically stable.
- Dpl 24-152 is soluble, sensitive to proteinase K, and exhibits biochemical properties akin to recombinant PrP.
Conclusions:
- Human Dpl shares biochemical similarities with PrP.
- Understanding Dpl's molecular features is crucial for future research on its function and role in neurodegenerative diseases.