Recombinant expression of soluble murine prion protein for C-terminal modification.

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.

Protein immobilization on liposomes and lipid-coated nanoparticles by protein trans-splicing.

A plethora of methods exist to link proteins to surfaces in order to generate functionalized materials. However, general tools that lead to functional immobilization of recombinantly expressed proteins on membranes such as liposomes or lipid-coated nanoparticles are rare. Here we present an approach that takes advantage of a double-palmitoylated peptide that mediates stable membrane anchoring in combination with protein trans-splicing for efficient immobilization of recombinant proteins fused to split intein segments. Two different DnaE split inteins from Synechocystis and Nostoc punctiforme are tested and compared to immobilization via direct native chemical ligation using a protein thioester. Protein trans-splicing proceeds at low protein concentrations and leads to functionalized vesicles and membrane-coated silica nanoparticles.

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.