Expression and structural characterization of the recombinant human doppelprotein

The doppel protein (Dpl) is a newly recognized prion protein (PrP)-like mol ecule encoded by a novel gene locus, prnd, located on the same chromosome a s 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 recombin ant Dpl 24-152 was characterized using gel electrophoresis, N-terminal Edma n sequencing, matrix-assisted laser desorption ionization mass spectrometry , and electrospray ionization mass spectrometry, Dpl 24-152 was shown to co ntain two disulfide bonds (Cys94-Cys145 and Cys108-Cys140). The secondary s tructure 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 denaturati on, In addition, Dpl was found to be soluble and sensitive to proteinase K digestion. Therefore, Dpl 24-152 possesses biochemical properties similar t o those of recombinant PrP. This study provides knowledge about the molecul ar features of human Dpl that will be useful in further investigation into its normal function and the role it may play in neurodegenerative diseases.