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Protein Engineering, Vol. 15, No. 6, 529-537, June 2002
© 2002 Oxford University Press

High-level expression and characterization of a glycosylated covalently linked dimer of the prion protein

Maria Louise Riley1,2, Christoph Leucht1, Sabine Gauczynski1, Christoph Hundt1, Martina Brecelj1, Guy Dodson2 and Stefan Weiss1,3

1 Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich, Germany and 2 National Institute for Medical Research, Mill Hill, Ridgeway, LondonNW7 1AA, UK

There is evidence that prion protein dimers may be involved in the formation of the scrapie prion protein, PrPSc, from its normal (cellular) form, PrPc. 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 PrPSc 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, PrPc/PrPSc conversion studies and other applications in the life cycle of prions.


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