Bovine Prion Protein Research Articles

Development of a nanoparticle-based surface-modified fluorescence assay for the detection of prion proteins

A nanoparticle-based immunoassay for the detection of recombinant bovine prion protein (PrP) was developed as a step in the development of screening tools for the prevention of the spread of transmissible spongiform encephalopathies. The assay is based on the competitive binding between PrP and a peptide–fluorophore to a nanoparticle-labeled antibody which is specific for a conserved prion sequence. The fluorophore, when bound to the antibody, is subject to surfaced-modified fluorescence, enabling detection of changes in the concentration of bound fluorophore in the presence of prion protein. Important factors considered during the development of the assay were ease of use, robustness, and detection level. The effects of pH and nanoparticle conjugation chemistry on surface-modified fluorescence observed in the assay were explored. Effects of concentrations of antibody and fluorophore on reproducibility and detection limits were examined. At present, the detection limits of the system are approximately equal to the antibody–peptide fluorophore equilibrium dissociation constant, which is near one nanomolar concentration. Improved assay performance could be obtained by optimization of the nanoparticle surface resonance effects. The simplicity of the assay and ease of use may make the type of assay described in this report attractive for screening purposes in the food industry.

Induction of Immune Response by Synthetic Fragments of the Bovine Prion Protein and Their Analogues in Mice of Various Lines

The antibodies to the bovine prion protein were produced by immunizing mice of three lines with five synthetic fragments of the protein and their six analogues. The analogues contained the amino acid substitutions that, according to theoretical calculation, should lead to an increase in the immunogenic activity of peptides. All the peptides, except for one, induced the formation of antibodies. All the sera containing the antipeptide antibodies were tested by an immunohistochemical method. The sera that were effectively bound to the brain preparations from the bovine with spongiform encephalopathy were identified; it was shown that they do not interact with the preparations of normal brain. Therefore, it was shown that the immunization of mice with the synthetic fragments of a prion protein helps obtain specific antibodies suitable for the study and diagnostics of prion diseases.

Sequential targeting of the genes encoding immunoglobulin-mu and prion protein in cattle.

Gene targeting is accomplished using embryonic stem cells in the mouse but has been successful, only using primary somatic cells followed by embryonic cloning, in other species. Gene targeting in somatic cells versus embryonic stem cells is a challenge; consequently, there are few reported successes and none include the targeting of transcriptionally silent genes or double targeting to produce homozygotes. Here, we report a sequential gene targeting system for primary fibroblast cells that we used to knock out both alleles of a silent gene, the bovine gene encoding immunoglobulin-mu (IGHM), and produce both heterozygous and homozygous knockout calves. We also carried out sequential knockout targeting of both alleles of a gene that is active in fibroblasts, encoding the bovine prion protein (PRNP), in the same genetic line to produce doubly homozygous knockout fetuses. The sequential gene targeting system we used alleviates the need for germline transmission for complex genetic modifications and should be broadly applicable to gene functional analysis and to biomedical and agricultural applications.

Purification of recombinant bovine normal prion protein PrP(104–242) by HPHIC

Purification of the prion protein (PrP) is a major concern for biological or biophysical analysis as are the structural specificities of this protein in relation to infectivity. A simple and efficient method for purification of recombinant bovine normal prion protein containing residues 104–242, PrP(104–242) expressed in Escherichia coli by high performance hydrophobic interaction chromatography (HPHIC) was presented in this work. The solution containing denatured and reduced protein in 8.0 mol/L urea extracted from the inclusion body was directly injected into the HPHIC column, aggregates were prevented by the interaction between the denatured PrP(104–242) molecules and the stationary phase during the chromatographic process, the soluble form of PrP(104–242) in aqueous solution was obtained after desorbed from the column. Several factors, including pH value, types of stationary phase and salt, and gradient mode, influencing the purification results were investigated. Optimal conditions were obtained for the purification of PrP(104–242) by HPHIC. This procedure yield PrP(104–242) of a purity of 96% with a recovery of 87%, respectively, for a single step purification of 40 min.

Different Behavior toward Bovine Spongiform Encephalopathy Infection of Bovine Prion Protein Transgenic Mice with One Extra Repeat Octapeptide Insert Mutation

In humans, insert mutations within the repetitive octapeptide region of the prion protein gene (Prnp) are often associated with familial spongiform encephalopathies. In this study, transgenic mice expressing bovine PrP (boTg mice) bearing an additional octapeptide insertion to the wild type (seven octapeptide repeats instead of six) showed an altered course of bovine spongiform encephalopathy (BSE) infection, reflected as reduced incubation times when compared with boTg mice expressing similar levels of the wild-type six-octapeptide protein. In both boTg mouse lines (bo6ORTg and bo7ORTg), incubation times were affected drastically depending on transgene expression levels and the inoculum used. In accordance with the lack of an interspecies barrier to BSE infection, we detected the typical signs of CNS spongiform degeneration by histopathological analysis and the presence of the bovine prion PrP(res) by Western blot or immunohistochemical analyses. When 7OR-PrP(res) was propagated in bo7ORTg mice, a similar earlier onset of clinical signs was observed compared with bo6ORTg mice. Proteins PrP(C) and PrP(res) containing seven octapeptides (7OR-PrP(C) and 7OR-PrP(res)) showed similar protease sensitivity and insolubility in nondenaturing detergents to homologous 6OR-PrP(C) and 6OR-PrP(res). In addition, bo7ORTg mice showed a higher sensitivity than bo6ORTg mice for detecting prion infection in specimens previously diagnosed as negative by conventional biochemical techniques. In the absence of clinical signs of disease, 7OR-PrP(res) could be detected as early as 120 d after inoculation by immunohistochemical and Western blot analyses. These findings may help us improve the current mouse bioassays and understand the role of the octapeptide repeat region in susceptibility to disease.

Inter- and Intra-octarepeat Cu(II) Site Geometries in the Prion Protein: IMPLICATIONS IN Cu(II) BINDING COOPERATIVITY AND Cu(II)-MEDIATED ASSEMBLIES

Cu(II) binding to the alpha prion protein (alphaPrP) can be both intramolecular and intermolecular. X-ray absorption spectroscopy at the copper K-edge has been used to explore the site geometry under each binding mode using both insoluble polymeric Cu(II).alphaBoPrP-(24-242) (bovine PrP) complexes and soluble Cu(II) complexes of peptides containing one, two, and four copies of the octarepeat. Analysis of the extended region of the spectra using a multiple scattering approach revealed two types of sites differing in the number of His residues in the first coordination shell of Cu(II). Peptides containing one and two-octarepeat copies in sub-stoichiometric Cu(II) complexes showed the direct binding of a single His in accord with crystallographic intra-repeat geometry. Alternatively, the polymeric Cu(II).alphaBoPrP-(24-242) complex and Cu(II) in its soluble complex with a four-octarepeat peptide at half-site-occupancy showed Cu(II) directly bound to two His residues, consistent with an inter-repeat binding mode. Increasing the Cu(II) site occupancy from 0.5 to 0.75 in the peptide containing four octarepeats resulted in spectral features that are intermediate to those of the inter- and intra-repeat modes. The transition from His-Cu-His (inter-repeat) to Cu-His (intra-repeat) on increasing Cu(II) saturation offers a structural basis for the positive cooperativity of the cation binding process and explains the capacity of alphaPrP to participate in Cu(II)-mediated intermolecular interactions.

An engineered PrPsc-like molecule from the chimera of mammalian prion protein and yeast Ure2p prion-inducing domain.

Production of the pathogenic prion isoform PrPsc-like molecules is thought to be useful for understanding the mysterious mechanism of conformational conversion process of prion diseases and proving the "protein-only" hypothesis. In this report, an engineered PrPsc-like conformation was produced from a chimera of mammalian bovine prion protein (bPrP) and yeast Ure2p prion-inducing domain (UPrD). Compared with the normal form of bPrP, the engineered recombinant protein, termed bPrP-UPrD, spontaneously aggregated into ordered fibrils under physiological condition, displaying amyloid-like characteristics, such as fibrillar morphology, birefringence upon binding to Congo red and increased fluorescence intensity with Thioflavine T. Limited resistance to protease K digestion and CD spectroscopy experiments suggested that the structure of bPrP-UPrD had been changed, and adopted a new, high content beta-sheet conformation during the fibrils formation. Moreover, bPrP-UPrD amyloid fibrils could recruit more soluble forms into the aggregates. Therefore, the engineered molecules could mimic significant behaviors of PrPsc and will be helpful for further understanding the mechanism of conformational conversion process.

Analysis of sequence variability of the bovine prion protein gene (PRNP) in German cattle breeds.

Different alleles of the prion protein gene (PRNP) of human and sheep are known to be associated with varying susceptibilities to transmissible spongiform encephalopathies. However, no polymorphisms in the bovine PRNP gene with an effect on susceptibility to prion diseases have been identified to date. In this study we investigated such polymorphisms in German cattle; 48 healthy animals from six different German cattle breeds and 43 cattle with bovine spongiform encephalopathy (BSE) were analyzed. In contrast to previous studies, all three exons as well as the promoter region of the PRNP gene were investigated. Sequence variants in the bovine PRNP gene could have an impact on the amino acid sequence or the expression level of the prion protein and thus on susceptibility to BSE. We identified a total of 60 polymorphisms in the PRNP gene of German cattle. Of these 60 polymorphisms, 36 were newly identified, whereas 24 of these polymorphisms had been described previously. We did not detect any novel polymorphisms affecting the amino acid sequence of the prion protein. However, we identified a 23-bp insertion/deletion polymorphism in the putative PRNP promoter region that shows a significant association with BSE susceptibility in our animals.

145COMBINED GLUCOSE AND FRUCTOSE SUPPLEMENTATION IN PROTEIN-FREE KSOM IMPROVES PREIMPLANTATION DEVELOPMENT OF BOVINE TRANSGENIC CLONED EMBRYOS

The present study investigated the effect of fructose supplementation in protein-free potassium simplex optimization medium (KSOM) on the preimplantation development of bovine transgenic cloned embryos. An expression plasmid containing bovine mutant PrP gene and enhanced green fluorescent protein (eGFP) as a marker gene was constructed and transfected into bovine fetal fibroblasts using FuGene6 (Roche, Indianapolis, IN, USA) as a lipid carrier. Transfected cells were cultured for 5 to 6 days to achieve chromosomal integration of the gene and then used for nuclear transfer. The somatic cell nuclear transfer was carried out by transferring a GFP-expressing donor cell into the perivitelline space of an enucleated oocyte. After electrical fusion and chemical activation, 525 (11 replicates) fused embryos were cultured in KSOM supplemented with 0.01% (w/v) PVA for 192 h at 39°C under 5% CO2, 5% O2 and 90% N2 gas atmosphere. The embryos were randomly allocated for 4 culture groups; KSOM supplemented with 1) 0.2mM glucose, 2) 1.5mM fructose, 3) 0.2mM glucose+1.5mM fructose and 4) vehicle (without glucose and fructose). We used 0.2mM glucose as per formulation of KSOM (Biggers JD et al. 2000 Biol. Reprod. 63, 281–293) and 1.5mM fructose due to its benefical effect on embryo development (Kwun J et al. 2003 Mol. Reprod. Dev. 65, 167–174). As a control experiment, 1043 (17 replicates) in vitro fertilized (IVF) embryos were cultured in the same culture system. The data were analyzed by PROC-GLM using SAS program. In IVF embryos, no significant differences in rates of cleavage (71.7 to 75.5%), morulae (34.1 to 37.1%) and blastocysts formation (21.0 to 24.5%) among the culture groups were observed. In contrast, significantly (P<0.05) higher rate in blastocysts formation (19.2%) was obtained when transgenic cloned embryos were cultured in KSOM supplemented with 0.2mM glucose+1.5mM fructose than with 0.2mM glucose (10.0%). However, the differences in blastocysts formation rates among other culture groups (13.2% for 1.5mM fructose and 11.9% for vehicle) were not significant. Moreover, the rates of cleavage (76.2 to 79.1%), morulae (19.3 to 23.8%) and GFP expression in blastocysts (68.0 to 78.6%) did not differ significantly among the culture groups for transgenic cloned embryos. This study demonstrated that the requirement of energy substrates in culture medium for bovine transgenic cloned and IVF embryos might be different. Now, we are conducting experiments to confirm whether the beneficial effect on transgenic cloned embryo development is due to combined effect of glucose and fructose or due to total concentration of available energy. This study was supported by Biogreen 21-1000520030100000.

1P090 蛍光相関分光法を用いた牛プリオン蛋白質の検出(蛋白質 E) 計測・解析の方法論)

1P090 蛍光相関分光法を用いた牛プリオン蛋白質の検出(蛋白質 E) 計測・解析の方法論)

Cu 2+ binding triggers αBoPrP assembly into insoluble laminar polymers

Cu 2+ binding is so far the best characterized property of the prion protein. This interaction has been mapped to the N-terminal domain of the prion protein where multiple His residues occur largely embedded within the repetitive PHGGGWGQ sequence known as octarepeats. When Cu 2+ interaction is studied using a solution of full-length bovine prion protein containing six octarepeats at protein concentrations above 25 μM, a drastic increase in solution turbidity is observed due to the formation of insoluble cation–protein complexes that appear as bidimensional polymer meshes. These bidimensional meshes consist of a single layer of protein molecules crosslinked by Cu 2+ cations. Polymer formation is a cooperative process that proceeds by nucleation of protein molecules with a Cu 2+ site occupancy of above 2. These results support the hypothesis that the N-terminal domain of prion protein is a ligand binding module that promotes crosslinked assembly, and suggest the existence of inter-repeat Cu 2+ sites.

Proteinase K enhanced immunoreactivity of the prion protein-specific monoclonal antibody 2A11

Here, we report the development and further characterisation of a novel PrP-specific monoclonal antibody: 2A11. By Western blot analysis, 2A11 reacts with PrP C from a variety of species including cow, sheep, pig, hamster, rabbit, cat, dog, deer and mouse but fails to react with human, chicken and turtle PrP. Reactivity to PrP C in Western blot was found to be dependent on the redox state of the protein since binding of mAb 2A11 to its epitope was more effective in reducing conditions. 2A11 binding site was mapped within a region comprised by residues 171–179 (six octarepeats bovine PrP notation; 163–171 for the ovine PrP notation). Interestingly, in immunohistochemistry (IHC) analysis, immunoreactivity was greatly enhanced after proteinase K (PK) sample treatment, while little or no reaction was observed in non-PK-treated BSE samples and samples from healthy animals. Quantitative differences in reactivity to BSE prions after PK treatment were also observed, to a lesser extent, by Western blot analysis. Since definitive diagnosis of prion diseases rely on IHC assays of proteinase K-treated samples, the use of mAb 2A11 might contribute to reduce the occurrence of false positive detection due to incomplete proteinase K digestion.

The hydrophobic internal region of bovine prion protein shares structural and functional properties with HIV type 1 fusion peptide.

The conserved fusion peptide at the N-terminus of HIV-1 envelope glycoprotein gp41 is involved in the virus-cell fusion reaction and in the cytopathic effects promoted by expression in single cells. The conserved bovine prion protein 121KHVAGAAAAGAVVGGLGGYMLGSAMSR147 transmembrane region (BPrP(tm)) contains a sequence rich in Gly residues [i.e., 130GAVVGGLGGYMLGSAMSR147 (BPrP(mi))] that shows homology with HIV-1 fusion peptide. As in the case of the latter peptide, analysis of the BPrP(mi) interfacial hydrophobicity confirms that this stretch bears an intrinsic tendency to partitioning from the aqueous phase into membranes. Experimental analyses of BPrP(mi)-lipid interactions suggest several similarities between this sequence and HIV-1 fusion peptide. Infrared spectroscopy reveals a conformational plasticity of the membrane-associated prion sequence comparable to that displayed by the viral sequence. The adoption of a mainly alpha-helical structure correlates with the formation of lytic pores. This helical structure can be converted into a beta-sheet conformation by addition of calcium, a process that is accompanied by vesicle membrane fusion. In contrast, transmembrane BPrP(tm) associates with membranes in a nonlytic, mainly helical structure but also containing some random coil. Upon addition of calcium, the random coils disappear while the helical conformation remains. In the absence of membranes both prion and HIV-1 sequences form amyloid-type fibers. It is proposed that during the pathological processes induced by secreted PrPSc and its proteolytic fragments, conformational polymorphism displayed by membrane-inserted BPrP(mi) may play a role at perturbing the general architecture of the membrane lipid bilayer and inducing protein-protein aggregation at membrane surfaces. These findings suggest the existence of common mechanisms underlying cytotoxicity by PrP and HIV-1 gp41.

Characterization of 2′-Fluoro-RNA Aptamers That Bind Preferentially to Disease-associated Conformations of Prion Protein and Inhibit Conversion

We have isolated artificial ligands or aptamers for infectious prions in order to investigate conformational aspects of prion pathogenesis. The aptamers are 2'-fluoro-modified RNA produced by in vitro selection from a large, randomized library. One of these ligands (aptamer SAF-93) had more than 10-fold higher affinity for PrPSc than for recombinant PrPC and inhibited the accumulation of PrPres in near physiological cell-free conversion assay. To understand the molecular basis of these properties and to distinguish specific from non-specific aptamer-PrP interactions, we studied deletion mutants of bovine PrP in denatured, alpha-helix-rich and beta-sheet-rich forms. We provide evidence that, like scrapie-associated fibrils (SAF), the beta-oligomer of PrP bound to SAF-93 with at least 10-fold higher affinity than did the alpha-form. This differential affinity could be explained by the existence of two binding sites within the PrP molecule. Site 1 lies within residues 23-110 in the unstructured N terminus and is a nonspecific RNA binding site found in all forms of PrP. The region between residue 90 and 110 forms a hinge region that is occluded in the alpha-rich form of PrP but becomes exposed in the denatured form of PrP. Site 2 lies in the region C-terminal of residue 110. This site is beta-sheet conformation-specific and is not recognized by control RNAs. Taken together, these data provide for the first time a specific ligand for a disease conformation-associated site in a region of PrP critical for conformational conversion. This aptamer could provide tools for the further analysis of the processes of PrP misfolding during prion disease and leads for the development of diagnostic and therapeutic approaches to TSEs.

On-column purification and refolding of recombinant bovine prion protein: using its octarepeat sequences as a natural affinity tag

Prion protein has a key role in the occurrence of transmissible spongiform encephalopathy (TSE) and development of these diseases. Here, we provide a convenient procedure for on-column purification and refolding of the full-length mature bovine prion protein (bPrP) from Escherichia coli using immobilized metal (Ni) affinity chromatography, based on the metal-binding property of its unusual octarepeat sequences containing six tandem copies. Following extensive washing, the bPrP pellet was solubilized by guanidine hydrochloride and subjected to Ni–NTA agarose column. Purification and refolding were achieved by stepwise gradient washing with reduced guanidine hydrochloride concentrations. Triton X-100 and β-mercaptoethanol were required in this rapid refolding process. The isolated prion protein was identified by monoclonal antibodies and its integrity was monitored by mass spectroscopy. Its correct folding was confirmed from circular dichroism (CD) experiments. Moreover, thioflavin T-binding assay showed that the recombinant bPrP could be transformed into amyloid fiber structures like that of the infectious prion isoform PrP sc.

Survey of cattle in northeast Colorado for evidence of chronic wasting disease: geographical and high-risk targeted sample.

A geographically targeted survey of potentially high-risk, adult cattle in chronic wasting disease (CWD)-endemic areas in Colorado was initiated to assess the possibility of the spread of CWD from deer to cattle under natural conditions. Surveyed cattle were sympatric with free-roaming deer in geographically defined areas where CWD occurs and where CWD prevalence has been estimated. To qualify for inclusion in the survey, cattle had to be at least 4 years old and had to have spent a minimum of 4 years in surveyed areas. Brains from culled cattle were examined microscopically and immunohistochemically for tissue alterations indicative of a transmissible spongiform encephalopathy (TSE). Two hundred sixty-two brains were suitable for evaluation and were found to lack changes indicative of a TSE infection. Prion deposition was not demonstrable using a method involving formic acid and proteinase-K treatment before application of monoclonal antibody to bovine prion protein (F99/97.6.1). Some incidental neuropathologic changes unrelated to those of TSEs were detected. Findings from this study suggest that large-scale spread of CWD from deer to cattle under natural range conditions in CWD-endemic areas of northeast Colorado is unlikely.

Intra- and interspecies interactions between prion proteins and effects of mutations and polymorphisms.

Recently, crystallization of the prion protein in a dimeric form was reported. Here we show that native soluble homogeneous FLAG-tagged prion proteins from hamster, man and cattle expressed in the baculovirus system are predominantly dimeric. The PrP/PrP interaction was confirmed in Semliki Forest virus-RNA transfected BHK cells co-expressing FLAG- and oligohistidine-tagged human PrP. The yeast two-hybrid system identified the octarepeat region and the C-terminal structured domain (aa90-aa230) of PrP as PrP/PrP interaction domains. Additional octarepeats identified in patients suffering from fCJD reduced (wtPrP versus PrP + 9OR) and completely abolished (PrP + 9OR versus PrP + 9OR) the PrP/PrP interaction in the yeast two-hybrid system. In contrast, the Met/Val polymorphism (aa129), the GSS mutation Pro102Leu and the FFI mutation Asp178Asn did not affect PrP/PrP interactions. Proof of interactions between human or sheep and bovine PrP, and sheep and human PrP, as well as lack of interactions between human or bovine PrP and hamster PrP suggest that interspecies PrP interaction studies in the yeast two-hybrid system may serve as a rapid pre-assay to investigate species barriers in prion diseases.

Susceptibility of Transgenic Mice Expressing Chimeric Sheep, Bovine and Human PrP Genes to Sheep Scrapie.

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.

BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein.

Variant Creutzfeldt-Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrP(Sc) type 2. These data suggest that more than one BSE-derived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.

Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance.

The Semliki-Forest virus (SFV) system was used to overexpress human wild-type and mutant prion proteins as well as FLAG-tagged human and bovine PrP in mammalian cells. The application of recombinant SFV vectors allowed a high-level production of highly glycosylated prion proteins with a molecular weight ranging from 25 to 30 kDa for recombinant wild-type human PrP and from 26 to 32 kDa for wild-type bovine PrP. Further, we report here the generation of recombinant mutant prion proteins that are associated with inherited human prion diseases such as fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Both mutated variants, the FFI-associated PrP carrying a mutation at amino acid position 178 and the CJD-linked form containing an insertion of nine additional octarepeats reveal proteinase K resistance, one of the typical biochemical properties of the infectious scrapie isoform of the prion protein. By contrast, recombinant wild-type PrP was completely proteinase K sensitive when expressed in SFV-transfected BHK cells. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP. In order to purify recombinant human and bovine PrP from cell lysates, a FLAG-tag was introduced either at the N-terminus behind the signal peptide or at the C-terminus close to the adhesion site of the GPI anchor. N-terminal insertion did not extensively influence the trafficking of the FLAG-tagged protein to the cell surface, whereas insertion close to the GPI attachment site clearly affected the transport of the majority of PrP to the cell membrane, probably resulting in their retention within the secretory pathway. All FLAG-tagged prion proteins were expressed efficiently in BHK cells and showed a typical glycosylation pattern, allowing their rapid and simple purification via anti-FLAG antibody chromatography.