High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein.
There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrP(Sc), from its normal (cellular) form, PrP(c). Recently, the crystal structure of the human prion protein in a dimeric form was reported. Here we report for the first time the overexpression of a human PrP dimer covalently linked by a FLAG peptide (PrP::FLAG::PrP) in the methylotrophic yeast Pichia pastoris. FLAG-tagged human PrP (aa1-aa253) (huPrP::FLAG) was also expressed in the same system. Treatment with tunicamycin and endoglycosidase H showed that both fusion proteins are expressed as various glycoforms. Both PrP proteins were completely digested by proteinase K (PK), suggesting that the proteins do not have a PrP(Sc) structure and are not infectious. Plasma membrane fractionation revealed that both proteins are transported to the plasma membrane of the cell. The glycosylated proteins might act as powerful tools for crystallization trials, PrP(c)/PrP(Sc) conversion studies and other applications in the life cycle of prions.
Riley
Maria Louise
ML
Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich, Germany.
Leucht
Christoph
C
Gauczynski
Sabine
S
Hundt
Christoph
C
Brecelj
Martina
M
Dodson
Guy
G
Weiss
Stefan
S
D019281:Dimerization; D019286:Endopeptidase K; D004765:Enteropeptidase / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010843:Pichia; D011233:Precipitin Tests; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014415:Tunicamycin / Q000378:metabolism
D009842:Oligopeptides; D010455:Peptides; D011328:Prions; D011993:Recombinant Fusion Proteins; D014415:Tunicamycin; C085706:FLAG peptide; D006026:Glycoside Hydrolases; D019286:Endopeptidase K; D004765:Enteropeptidase