title: Recombinant expression of soluble murine prion protein for C-terminal modification.
abstract: Membrane attachment of prion protein (PrP) via its glycosylphosphatidylinositol (GPI) anchor plays a key role during conversion of cellular PrP(C) into its pathogenic isoform PrP(Sc). Strategies to access homogenous lipidated PrP via expressed protein ligation (EPL) are required to fully decipher the effect of membrane attachment. Such strategies suffer from insoluble expression of PrP-intein fusion constructs and low folding efficiencies that severely limit the available amount of homogeneous lipidated PrP. Here, we describe an alternative method for expression of soluble PrP-intein fusion proteins in Escherichia coli that provides access to natively folded PrP ready to use in EPL.
authors: Institute of Biological Chemistry, University of Vienna, Vienna, Austria.
authors: Nam Ky
authors: NK
authors: Chu
authors: Christian F W
authors: CF
authors: Becker
mesh_terms: D000818:Animals; D001665:Binding Sites; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000737:chemistry; D004926:Escherichia coli; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D005456:Fluorescent Dyes / Q000737:chemistry; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D047668:Inteins; D051379:Mice; D011328:Prions / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D018840:HSP70 Heat-Shock Proteins; D011328:Prions; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; C099488:TEV protease; C063842:dnaK protein, E coli