Semisynthesis of membrane-attached prion proteins.
Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) is the hallmark of prion diseases and has been studied extensively by using recombinantly expressed PrP (rPrP). Because of the inherent difficulties of expressing and purifying posttranslationally modified rPrP variants only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Protein semisynthesis provides two alternative routes to access multimilligram amounts of membrane-attached rPrP, which are described in detail here. In both cases, rPrP fused to a C-terminal extension comprising either the Mycobacterium xenopi GyrA mini-intein or the Synechocystis sp. DnaE N-terminal split intein is expressed in E. coli. Protein purification was followed by reaction with chemically synthesized palmitoylated membrane anchor peptides to yield rPrP(Palm) or with a chemically synthesized glycosylphosphatidylinositol (GPI) anchor to give rPrP(GPI). Solubility problems encountered with synthetic membrane anchors were overcome by either incorporating a polyethylene glycol-based C-terminal tag (removable by specific proteolysis) or by direct incorporation into liposomes. The new rPrP(Palm) variants studied by a variety of in vitro methods exhibited a high affinity to liposomes and an increased lag phase during aggregation when compared to rPrP. Similar results were obtained for rPrP(GPI), in which only one alkyl chain is sufficient for quantitative membrane attachment. In vivo studies demonstrated that double lipidated rPrP(Palm) is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells.
Chu
Nam Ky
NK
Technische Universität München, Department of Chemistry, Protein Chemistry Group, Garching, Germany.
Becker
Christian F W
CF
D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006801:Humans; D047668:Inteins; D008081:Liposomes / Q000378:metabolism; D054878:Lipoylation; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D019308:Palmitic Acid / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis* / Q000378:metabolism; D018621:PrPC Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D019154:Protein Splicing; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification
D017261:Glycosylphosphatidylinositols; D008081:Liposomes; D008565:Membrane Proteins; D010455:Peptides; D018621:PrPC Proteins; D011993:Recombinant Fusion Proteins; D019308:Palmitic Acid