Expression and structural characterization of the recombinant human doppel protein.
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.
Lu
K
K
Institute of Pathology and Mass Spectrometry Core Facility, Case Western Reserve University, 2085 Adelbert Road, Cleveland, Ohio 44106, USA.
Wang
W
W
Xie
Z
Z
Wong
B S
BS
Li
R
R
Petersen
R B
RB
Sy
M S
MS
Chen
S G
SG
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D058851:GPI-Linked Proteins; D006801:Humans; D006868:Hydrolysis; D007106:Immune Sera / Q000096:biosynthesis / Q000737:chemistry; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011328:Prions / Q000737:chemistry* / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification; D012995:Solubility
D058851:GPI-Linked Proteins; D007106:Immune Sera; C501125:PRND protein, human; D011328:Prions; D011994:Recombinant Proteins; D010450:Endopeptidases