In vitro cell-free conversion of bacterial recombinant PrP to PrPres as a model for conversion

Louise Kirby1, Christopher R. Birkett1, Helene Rudyk2, Ian H. Gilbert2 and James Hope1,{dagger}

1 Institute for Animal Health, Compton Laboratories, Newbury, Berkshire RG20 7NN, UK
2 Welsh School of Pharmacy, Cardiff University, Redwood Building, King Edward VII Avenue, Cardiff CF10 3XF, UK

Correspondence
Louise Kirby
louise.kirby{at}bbsrc.ac.uk


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Prion diseases are associated with the conversion of the normal cellular prion protein, PrPC, to the abnormal disease-associated protein, PrPSc. This conversion can be mimicked in vitro using PrPSc isolated from the brains of scrapie-infected animals to induce conversion of recombinant PrPC into a proteinase K-resistant isoform, PrPres. Traditionally, the ‘cell-free’ conversion assay has used, as substrate, recombinant PrPC purified from mammalian tissue culture cells or, more recently, from baculovirus-infected insect cells. The cell-free conversion assay has been modified by replacing the tissue culture-derived PrPC with recombinant PrP purified from bacteria. Bacterial expression and chromatographic purification give high yields of recombinant radiolabelled untagged protein, eliminates artefacts that may be due to cellular factors or antibody fragments normally present in labelled PrP preparations and allows accurate and rapid variation of protein sequence using standard molecular biological techniques. In addition, these cell-free conversion assays were carried out under more physiological conditions, giving more relevance to the assay as a model for conversion. To validate its use in this assay, this bacterial recombinant PrP has been shown to have the conversion properties of mammalian PrPC: (i) it converts to a proteinase K-resistant isoform in the presence of PrPSc; (ii) the efficiency of this conversion by PrPSc of different strains and species parallels that found in vivo; and (iii) its cell-free conversion is inhibited by Congo Red analogues in a structure-dependent manner similar to that seen in in vivo and in vitro cell assays.

Published ahead of print on 24 January 2003 as DOI 10.1099/vir.0.18903-0.

{dagger}Present address: VLA Lasswade, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 OPZ, UK.


   INTRODUCTION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Transmissible spongiform encephalopathies (TSEs) are fatal, neurodegenerative diseases of humans and animals and include Creutzfeldt–Jakob disease (CJD), Gerstmann–Straussler–Scheinker syndrome, Kuru, bovine spongiform encephalopathy (BSE), scrapie, transmissible mink encephalopathy and chronic wasting disease. At present, there is no treatment or cure.

The causative agent responsible for TSEs or prion diseases has yet to be fully defined. A fundamental event in disease is the conversion of the normal, detergent-soluble, proteinase K-sensitive isoform of the prion protein, PrPC, to an abnormal, detergent-insoluble, partially proteinase K-resistant isoform, PrPSc, and the accumulation of this abnormal isoform in the central nervous system of infected animals (Hope et al., 1986; Meyer et al., 1986; Oesch et al., 1985). The protein-only hypothesis suggested that no nucleic acid is needed for replication (Griffith, 1967); this has been refined by defining PrPSc as the infectious agent and replication as the conversion by PrPSc of host PrPC to more PrPSc (Prusiner et al., 1982). Although the mechanism of conversion is unknown, interaction between PrPC and PrPSc is critically implicated by in vivo studies (Caughey & Chesebro, 1997) and features in the current models of replication such as template-assisted (Prusiner, 1991) and nucleation-dependent (Jarrett & Lansbury, 1993) conversion.

The in vitro cell-free conversion assay has provided a quick, simple and well-defined system in which to study the molecular factors that influence the transition of PrPC to PrPSc (Kocisko et al., 1994). In this system, PrPSc, isolated from the brains of infected animals, induces the conversion of radiolabelled PrPC to a proteinase K-resistant isoform, PrPres. Newly formed PrPres is distinguished from the PrPSc used to seed the conversion by the fact that it is radiolabelled. The assay has been shown to replicate in vivo species specificity, strain properties and polymorphism barriers (Bessen et al., 1995; Bossers et al., 1997, 2000; Iniguez et al., 2000; Kocisko et al., 1995; Zhang et al., 2002; Raymond et al., 1997; Horiuchi et al., 2000) but as yet no in vitro-generated PrPres has been shown to be infectious (Hill et al., 1999).

Traditionally, cell-free conversion assays have used, as substrate, PrPC purified from mammalian tissue culture cells (Kocisko et al., 1994; Bossers et al., 2000; DebBurman et al., 1997; Saborio et al., 1999; Hill et al., 1999) or, more recently, from baculovirus-infected insect cells (Iniguez et al., 2000; Zhang et al., 2002). In this study, we have modified the cell-free conversion assay by replacing the tissue culture-derived PrPC with PrP purified from bacteria and refolded in vitro. In addition, the guanidine conversion buffer, usually required for efficient conversion in vitro, has been replaced with a conversion buffer approximating physiological conditions, similar to that used by Horiuchi et al. (1999).

We found that PrP derived from bacteria converts to a proteinase K-resistant isoform in the cell-free conversion assay and the assay was shown to mimic the in vivo species barriers of transmission of 263K hamster and 87V mouse scrapie between hamster and mouse.

With the emergence of BSE (Wells et al., 1987) and variant CJD (Will et al., 1996), the search for prophylactic and therapeutic compounds is under way. We investigated the use of our modified cell-free conversion assay for identifying compounds that inhibit this conversion. Ten compounds with known anti-TSE activity in cell culture and/or in vivo were tested for their ability to inhibit this conversion. Eight of these compounds inhibited cell-free conversion to some extent, suggesting part of their in vivo effect could be modulated by binding directly to PrP. Two compounds did not inhibit in vitro conversion; these compounds may inhibit in vivo conversion indirectly by acting on some other part of cell metabolism.

These studies reinforce the specificity of PrPC and PrPSc interactions and the utility of the cell-free conversion assay using bacterial recombinant PrP to investigate TSE transmission barriers and identify potential prophylactic compounds.


   METHODS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression, radiolabelling and purification of recombinant PrP.
Full-length mouse PrP of the Prn-pa genotype (Locht et al., 1986) with the N-terminal signal sequence replaced with methionine and the C-terminal signal sequence removed (corresponding to aa 23–230) was amplified from genomic DNA using the 5' and 3' primers 5'-GGGATCCATCATGAAAAAGCGGCCAAAGCCTGGAG-3' and 5'-CGAATTCTTAGGATCTTCTCCCGTCGTAATAG-3', respectively. Full-length hamster PrP (Basler et al., 1986) with the N-terminal signal sequence replaced with methionine and the C-terminal signal sequence removed (corresponding to aa 23–231) was amplified from genomic DNA using the 5' and 3' primers 5'-GGGATCCATCATGAAGAAGCGGCCAAAGCCT-3' and 5'-CGAATTCTCAGGACCTTCTTCC-3', respectively. Plasmid pTrcHis (Invitrogen) was digested with the restriction enzymes NcoI/EcoRI to remove the 6-histidine tag. PCR fragments were digested with restriction enzymes EcoRI/RcaI and ligated into the modified pTrcHis vector. Therefore, the recombinant vector encodes full-length, untagged PrP.

Calcium chloride-competent Escherichia coli strain 1B392 were transformed with the recombinant vectors. Cells were grown in 50 ml volumes in methionine-deficient M63 media containing ampicillin to an OD600 of approximately 0·2, induced with 1 mM IPTG and proteins labelled with 18·5 MBq of [35S]methionine for 90 min. Cells were harvested by centrifugation, lysed in buffer [50 mM Tris/HCl (pH 8), 100 mM sodium chloride and 1 mM EDTA] containing PMSF (0·1 mM final) and lysozyme (20 µg ml-1 final) and then treated with sodium deoxycholate (1 mg ml-1 final) and DNase (5 µg ml-1 final). Inclusion bodies were isolated by centrifugation and solubilized in 8 M urea. Insoluble material was removed by centrifugation and PrP in the supernatant purified in a two-stage chromatographic procedure of Ni-NTA affinity chromatography (Qiagen), binding in 0·1 M sodium phosphate, 10 mM Tris (pH 8), 8 M urea and 10 mM {beta}-mercaptoethanol and eluting in 0·1 M sodium phosphate, 10 mM Tris (pH 4·5), 8 M urea and 10 mM {beta}-mercaptoethanol, followed by cation-exchange chromatography using SP–Sepharose (Pharmacia), binding in 8 M urea and 50 mM HEPES (pH 8) and eluting with a sodium chloride gradient. Purification of PrP was followed by Western blotting using the monoclonal antibody (mAb) 6H4 (Prionics).

Following purification, 35S-labelled PrP was refolded by copper oxidation of the disulphide bond (Jackson et al., 1999) and dialysis into 50 mM sodium acetate (pH 5·5). Estimates of PrP purity were made by Coomassie staining and estimates of concentration were made using dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA, Perkin-Elmer) using unlabelled recombinant PrP as a standard, the mAb FH11 (TSE Resource Centre, I.A.H., Compton, UK) for capture of PrP onto a 96-well plate and europium-labelled mAb 6H4 for subsequent detection.

PrPSc preparation.
PrPSc was prepared from the brains of terminally ill 87V-infected VM mice and 263K-infected hamsters, based on a method described by Hope et al. (1986). Briefly, a 5 % (w/v) brain homogenate was prepared in 10 mM sodium phosphate (pH 7·4) and 10 % (w/v) N-lauryl sarcosinate. The suspension was centrifuged at 22 000 g for 30 min at 10 °C. The supernatant was centrifuged at 215 000 g for 150 min at 10 °C and the pellet resuspended in H2O. The volume of the solution was adjusted to 9 ml g-1 of brain and its ionic composition to 0·6 M potassium iodide, 6 mM sodium thiosulphate, 1 % (w/v) N-lauryl sarcosinate and 10 mM sodium phosphate (pH 8·5) and centrifuged at 285 000 g for 90 min at 10 °C through a sucrose cushion of 20 % (w/v) sucrose in 0·6 M potassium iodide, 6 mM sodium thiosulphate, 1 % (w/v) N-lauryl sarcosinate and 10 mM sodium phosphate (pH 8·5). The pellet was washed in H2O and centrifuged in a microfuge. The final PrPSc pellet was resuspended in H2O by sonication to approximately 1 µg µl-1.

Cell-free conversion assay.
Two cell-free conversion protocols were used in this study, either with or without guanidine. The cell-free conversion assay without guanidine was based on that used by Horiuchi et al. (1999). Briefly, PrPSc was sonicated and approximately 1 µg incubated with 200 ng of [35S]PrP in conversion buffer [50 mM citrate (pH 6·5), 50 mM potassium chloride, 10 mM magnesium chloride, 100 mM sodium chloride and 0·1 % (v/v) Nonidet P-40], either with or without compounds to be tested for inhibition, for 24 h at 37 °C in a 20 µl volume reaction. Following incubation, 20 µl H2O was added. One-twentieth of the reaction mixture was removed for analysis without proteinase K treatment and the rest was treated with 60 µg proteinase K ml-1 for 1 h at 37 °C. Proteinase K digestion was stopped by the addition of Pefabloc to 1 mM. All samples were precipitated with 20 µg BSA and 4 vols ice-cold methanol at -20 °C. The resulting pellet was boiled for 10 min in SDS-PAGE sample buffer and analysed by SDS-PAGE in 15 % polyacrylamide gels. Gels were then fixed, dried and exposed to film. Autoradiographs were quantified using Phoretix Gel Analysis software. For cell-free conversion assays in guanidine buffers, we based our method on that described in Kocisko et al. (1994). PrPSc was sonicated briefly and approximately 1 µg pre-incubated in 2 M guanidine, 0·25 % (w/v) zwittergent 3-14 and 50 mM citrate (pH 6·5) in a reaction volume of 10 µl for 2 h at 37 °C. Following pre-incubation, 200 ng [35S]PrP was added to give a final guanidine concentration of 1 M. The reaction was incubated for 24 h at 37 °C and proteinase K-treated as described above.

Compounds.
Compounds were obtained from Sigma-Aldrich or Lancaster or synthesized directly. Congo Red (CR) and its analogues were stored as 10x stock solutions in 2 % (v/v) DMSO at -20 °C. Chlorpromazine and quinacrine were freshly prepared as 10x stock solutions in H2O for each experiment. For compound structures, see Fig. 4.



View larger version (23K):
[in this window]
[in a new window]
 
Fig. 4. Chemical structures of inhibitor compounds. CR and related compounds (roman numerals) and chlorpromazine and quinacrine were screened for their ability to inhibit conversion of [35S]MoPrP to [35S]MoPrPres in the cell-free conversion assay.

 

   RESULTS
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Production of 35S-labelled PrP in recombinant bacteria
Recombinant mouse PrP (aa 23–230) was expressed in bacteria from the modified pTrcHis expression vector in the presence of [35S]methionine. Following purification and refolding in vitro, the recombinant protein was analysed by Western blotting with anti-PrP mAb (Fig. 1, lane 1) and Coomassie staining (Fig. 1, lane 2). The protein had a molecular mass of approximately 26 kDa, as expected for unglycosylated PrP lacking the glycosylphosphatidylinositol (GPI)-anchor addition peptide. The yield was approximately 100 µg [35S]MoPrP (recombinant mouse PrP) per 50 ml of culture medium and purity was estimated by Coomassie staining to be approximately 95 %. Mass spectrometry and circular dichroism spectroscopic analysis of unlabelled protein confirmed the correct expression, formation of the disulphide bond and folding into a normal {alpha}-helical formation (data not shown).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Western blot and Coomassie-stained gel analysis of [35S]MoPrP. Recombinant MoPrP was radiolabelled during expression from bacteria, purified and refolded in vitro. Western blot using mAb 6H4 (lane 1) and Coomassie-stained gel (lane 2) of purified refolded [35S]MoPrP. Molecular mass markers (kDa) are indicated on the left.

 
Conversion of bacterial PrP to PrPres
PrPC purified from mammalian tissue culture cells (Kocisko et al., 1994; Bossers et al., 2000; DebBurman et al., 1997; Saborio et al., 1999; Hill et al., 1999) and from baculovirus-infected insect cells (Zhang et al., 2002; Iniguez et al., 2000) has been used as substrate in the cell-free conversion assay. In this study, recombinant PrP, purified from bacterial cells and refolded in vitro, could also be converted into a proteinase K-resistant form in the cell-free conversion assay. Recombinant mouse PrP, MoPrP, was radiolabelled during expression, purified from bacteria and refolded in vitro. [35S]MoPrP was incubated with and without unlabelled PrPSc, isolated from 87V scrapie-infected VM mouse brains in a guanidine-free conversion buffer at 37 °C for 24 h. Fig. 2 (lanes 1 and 3) show undigested [35S]MoPrP. Following proteinase K digestion, [35S]MoPrP became more proteinase K resistant in the presence (Fig. 2, lane 4) but not in the absence (Fig. 2, lane 2) of PrPSc. The proteinase K-resistant core of [35S]MoPrPres was approximately 17 kDa, 6–7 kDa smaller than [35S]MoPrP, characteristic of the proteinase K-resistant core of PrPSc, PrP27-30 (Oesch et al., 1985; Hope et al., 1986). The efficiency of the conversion was approximately 30 %, according to densitometric analysis of labelled PrP before and after proteinase K treatment. This result indicates that recombinant PrP expressed and purified from bacteria and refolded in vitro is a substrate in the cell-free conversion assay under physiologically compatible conditions and conversion to PrPres is PrPSc dependent.



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 2. Cell-free conversion of recombinant bacterially expressed PrP. [35S]MoPrP was incubated with and without unlabelled 87V fibrils in non-guanidine conversion buffer at 37 °C for 24 h followed by proteinase K (PK) digestion. [35S]MoPrPres was detected by autoradiography. Lanes 1 and 3 show undigested [35S]MoPrP. [35S]MoPrPres was detected in the presence (lane 4) but not in the absence (lane 2) of PrPSc. Molecular mass markers (kDa) are indicated on the left.

 
[35S]MoPrP of the Prn-pa allotype was converted to PrPres by 87V (VM)-PrPSc of the Prn-pb allotype (Fig. 2), although, in vivo, mice with the Prn-pa genotype rarely if ever develop neurological disease following exposure to the 87V (VM) strain of mouse scrapie (M. E. Bruce, IAH, Edinburgh, UK, personal communication). However, this is only an apparent breakdown in the correlation between the cell-free conversion efficiency and in vivo transmissibility, since, while Prn-pa allotype mice rarely develop clinical signs, 87V scrapie readily replicates in their brains and spleens. Furthermore, pathology typical of brain scrapie has been found in some older mice dying of ‘natural causes’, suggesting that 87V-infected Prn-pa mice have prolonged survival times rather than a resistance to disease (M. E. Bruce, IAH, Edinburgh, UK, personal communication).

Species specificity in the conversion reaction
87V mouse scrapie has a prolonged incubation time in hamsters and mice are highly resistant to infection with 263K hamster scrapie. To determine whether our cell-free conversion assay mimics this in vivo species specificity, hamster and mouse PrP were radiolabelled and purified from the bacterial expression system, refolded in vitro and incubated in a non-guanidine conversion buffer, with PrPSc isolated from 263K-infected hamster brains and 87V-infected mouse brains in homologous and heterologous conversion assays. Hamster 35S-labelled PrP, [35S]HaPrP, was converted by hamster PrPSc into [35S]HaPrPres (Fig. 3, lane 2) and [35S]MoPrP was converted by mouse PrPSc into [35S]MoPrPres (Fig. 3, lane 6). Very little or no 17 kDa [35S]PrPres was produced when [35S]HaPrP was incubated with mouse PrPSc (Fig. 3, lane 4) or when [35S]MoPrP was incubated with hamster PrPSc (Fig. 3, lane 8). These data indicate that the species specificity observed in vivo can be mimicked in our cell-free conversion assay using recombinant bacterial PrP.



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 3. Species specificity in the cell-free conversion assay. [35S]PrP and PrPSc preparations were mixed and incubated in non-guanidine conversion buffer at 37 °C for 24 h. Following proteinase K (PK) digestion, [35S]PrPres was detected by autoradiography in the homologous conversion reactions (lane 2 and 6) but very little or no [35S]PrPres was observed in the heterologous conversion reactions (lane 4 and 8). These data are representative of three independent experiments. Molecular mass markers (kDa) are shown on the left. Ha, Hamster; Mo, mouse.

 
Inhibition of the cell-free conversion assay
There are several lead anti-TSE compounds, including CR, chlorpromazine and quinacrine (reviewed by Gilbert & Rudyk, 1999). CR inhibits PrPres formation in scrapie-infected mouse brain cells (SMB) (Rudyk et al., 2000), in the cell-free conversion assay and in scrapie-infected neuroblastoma cells (ScN2a) (Demaimay et al., 1998, 2000; Caughey & Race, 1992; Caughey et al., 1993) and can cure these cells of infectivity. CR also prolongs the survival time of scrapie-infected hamsters if administered around the time of experimental infection (Ingrosso et al., 1995). Although CR has anti-TSE activity, it has poor blood–brain barrier permeability and is toxic when broken down in the gut by microbes (Boss et al., 1987); therefore, its use as a drug is limited and structure activity studies have been carried out to improve its therapeutic properties (Rudyk et al., 2000; Demaimay et al., 1998). Chlorpromazine and quinacrine have been used in humans for many years as anti-psychotic and anti-malarial drugs, respectively, and have been shown to inhibit PrPres formation in ScN2a cells (Korth et al., 2001; Doh-Ura et al., 2000). Although these drugs have anti-TSE activity, their mechanisms of action are unknown.

We investigated the use of our cell-free conversion assay, using bacterial PrP, to study the inhibitory effect of such anti-TSE compounds. CR and its analogues and chlorpromazine and quinacrine (Fig. 4) were tested for their ability to inhibit formation of PrPres in our non-guanidine cell-free conversion assay using recombinant PrP purified from bacteria and refolded in vitro. [35S]MoPrP was incubated with 87V PrPSc with varying amounts of inhibitor compounds in the cell-free conversion assay. Fig. 5 shows a typical autoradiograph of inhibition of conversion of PrP to PrPres by CR and compound XXIV, Sirius Red (SR). CR and its analogues all inhibited conversion of [35S]MoPrP to PrPres to some extent (Fig. 5). SR was the most effective inhibitor, with an average IC50 of 6·2±0·26 µM. This was followed by CR, with an average IC50 of 8·3±0·35 µM, then by compounds XVIII, VI, IX, XVI and XII, with average IC50 titres ranging from 25 to 70 µM (Fig. 6). Compound III was the least effective inhibitor, with an average IC50 of 400 µM. As has been reported elsewhere (Demaimay et al., 1998, 2000; Rudyk et al., 2000) that at low concentrations, CR and its analogues enhanced conversion above the control level. Chlorpromazine and quinacrine did not inhibit conversion at concentrations of 0·1–100 µM in our cell-free conversion assay (data not shown).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 5. Dose response relationships for (a) CR and (b) compound XXIV, SR, in the cell-free conversion assay. [35S]MoPrP was incubated with 87V fibrils in non-guanidine conversion buffer at 37 °C for 24 h with various concentrations of inhibitor. Amount of [35S]MoPrPres in the absence of inhibitor is shown in lane 2. Lanes 3–7 indicate the amount of [35S]MoPrPres in the presence of increasing concentrations of inhibitor. Molecular mass markers (kDa) are shown on the left. PK, Proteinase K. (c, d) Representation of three independent experiments. Results are plotted as the percentage of [35S]MoPrPres generated in the cell-free conversion assay in the presence of compounds relative to the amount of PrPres generated in the absence of compounds, as determined by densitometry.

 


View larger version (8K):
[in this window]
[in a new window]
 
Fig. 6. Average IC50 titres for eight compounds that inhibited the formation of [35S]MoPrPres in the cell-free conversion assay. [35S]MoPrP was incubated with 87V fibrils in non-guanidine conversion buffer for 24 h at 37 °C with various concentrations of compounds. Following proteinase K treatment and SDS-PAGE, [35S]MoPrPres was detected by autoradiography. Average IC50 titres ±SE were calculated from dose response curves from three independent experiments.

 
These data indicate that CR and its analogues may inhibit in vivo conversion by binding directly to PrP, whereas chlorpromazine and quinacrine may inhibit in vivo conversion not by binding to PrP but by influencing some other event in the cell.

Guanidine prevents the inhibitory effect of CR
Previously, the inhibitory effects of CR and its analogues on the cell-free conversion of recombinant PrPC were investigated using protocols that incorporated guanidine (Demaimay et al., 1998, 2000). Although CR inhibits conversion of PrPC to PrPres under such conditions, we wanted to measure the effect of CR on conversion without contributions from such non-physiological salts. The efficiency of conversion of [35S]PrP was similar in both the presence and the absence of guanidine (Fig. 5a, lane 2, and Fig. 7, lane 2, respectively). However, when our more physiologically compatible conversion buffer was substituted with conversion buffer containing guanidine, CR no longer inhibited cell-free conversion at similar concentrations (Fig. 7).



View larger version (48K):
[in this window]
[in a new window]
 
Fig. 7. Dose response relationship for CR in a cell-free conversion assay containing guanidine. 87V fibrils were pre-incubated in 2 M guanidine for 1 h at 37 °C followed by dilution to 1 M guanidine by the addition of [35S]MoPrP and various concentrations of inhibitor and incubated at 37 °C for 24 h. Amount of [35S]MoPrPres in the absence of inhibitor is shown in lane 2. Lanes 3–7 indicate the amount of [35S]MoPrPres in the presence of increasing concentrations of CR. Molecular mass markers (kDa) are shown on the left. PK, Proteinase K.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Kocisko et al. (1994) were the first to demonstrate that PrPSc could induce the conversion of PrPC into a proteinase K-resistant isoform, PrPres, a hallmark of the disease-specific prion protein, in an in vitro cell-free assay and that this PrPres was capable of limited self propagation. Their study used recombinant PrPC immunoprecipitated from mammalian cell culture as the substrate for conversion (Kocisko et al., 1994) and although PrPC and PrPSc are the main constituents of the cell-free conversion assay, it is possible that cellular factors or antibody fragments introduced during the immunoprecipitation may co-purify with PrPC and influence the cell-free conversion assay; chaperones (DebBurman et al., 1997), metal ions (McKenzie et al., 1998) and cell lysates (Saborio et al., 1999) have all been shown to enhance conversion. In addition, the production of eukaryotic protein is time consuming and requires large amounts of radioactivity for low yields of radiolabelled protein.

Recently, recombinant PrP isolated either by immunoprecipitation (Iniguez et al., 2000) or using an histidine tag (Zhang et al., 2002) from baculovirus-infected insect cells has been used as a substrate in the cell-free conversion assay. These studies indicate that a non-mammalian source of PrP can be converted into a proteinase K-resistant species. In this study, we have used recombinant PrP expressed and purified from bacteria and refolded in vitro as a substrate in the cell-free conversion assay. This system uses relatively low amounts of radioactivity to produce high yields of recombinant protein, devoid of either an epitope tag or an histidine tag that may influence conversion, and because the protein is purified biochemically, complications such as the potential co-purification of cellular factors or antibody fragments is reduced. In addition, it is a quick and simple procedure and can allow rapid variation of protein sequence using standard cloning and mutagenesis techniques. Full-length mouse PrP (aa 23-230) was expressed, radiolabelled and purified from bacteria and refolded in vitro. The expressed protein had a molecular mass of approximately 26 kDa, characteristic of full-length, aglycosyl PrP lacking the GPI anchor. Kocisko et al. (1994) have demonstrated that PrP does not require glycosylation or a GPI anchor to be converted in their cell-free conversion assays. We were able to show that bacterial recombinant PrP can be converted into PrPres in the cell-free conversion assay with efficiencies of conversion similar to those obtained with mammalian and baculovirus recombinant PrP. The most abundant [35S]PrPres product of the cell-free conversion assay is 17 kDa, 6–7 kDa smaller than the precursor [35S]PrP. This is similar to the 6–7 kDa reduction in mass seen upon digestion of brain-derived PrPSc. Proteinase K-resistant species smaller than 17 kDa were frequently observed in cell-free conversion assays. These smaller species do not correlate with in vivo transmissibility and may represent by-products of a non-pathogenic folding pathway.

To validate this bacterial PrP conversion assay, we investigated if the in vivo transmissibilities of scrapie between hamster and mouse could be replicated in this assay. The barriers of transmission of TSEs from one species to another generally involve a prolonged incubation period. The species specificity observed in vivo has been reproduced in the cell-free conversion assay using mammalian (Kocisko et al., 1995; Raymond et al., 1997; Horiuchi et al., 2000) and baculovirus (Iniguez et al., 2000; Zhang et al., 2002) recombinant PrP. In this study, the results of conversion reactions between recombinant hamster and mouse PrP, and PrPSc of 263K hamster and 87V mouse, correlates with the relative transmissibility of scrapie between those species in vivo. Although molecular compatibility between PrPC and PrPSc is important in the transmission of TSEs, other factors such as dose, route of infection and strain of agent may influence conversion in vivo.

Chlorpromazine, quinacrine and CR (and its analogues) are known inhibitors of PrPres formation in tissue culture cells (Korth et al., 2001; Demaimay et al., 1998, 2000; Rudyk et al., 2000; Caughey & Race, 1992; Caughey et al., 1993) and have shown limited success in vivo (Ingrosso et al., 1995). We observed similarities and differences in the ability of these compounds to inhibit conversion in the bacterial PrP cell-free assay. Chlorpromazine and quinacrine, contrary to their inhibitory effects on PrPres formation in ScN2a cells (Korth et al., 2001), did not inhibit PrPres formation in the cell-free conversion assay. This suggests that in vivo chlorpromazine and quinacrine inhibit conversion not by binding directly to PrP but in some less direct effect on the cell. A recent study investigating the efficacy of quinacrine in an in vivo model of mouse-adapted scrapie failed to show a significant increase in survival time of scrapie-infected mice following quinacrine administration (Collins et al., 2002). Alternatively, this lack of effect in the cell-free conversion assay may be due to host and TSE strain variables that are known to influence the effectiveness of anti-TSE drugs in vivo. The CR analogues used in our cell-free conversion assay were screened previously for their ability to inhibit PrPres formation in SMB cells (Rudyk et al., 2000). Although the IC50 titres reported in the cellular assay were not identical to the titres generated in the cell-free system, the order of effectiveness as inhibitors was the same. In summary, SR was the most potent inhibitor in both assays. The half molecule of CR had some activity but only at high concentrations. Compounds with an increased level of sulphation, substitution of the naphthelene amino with trifluoroacetamide, 3,3'-modification of the biphenyl or replacement of the biphenyl with bisulphone, all retained some activity. This suggests that it is possible to modify the structure of CR without dramatically affecting its activity as an inhibitor and therefore, it should be possible to design a compound with improved pharmacokinetic properties. The correlation between the cell-free and the cellular conversion assays suggests that in vivo CR may prevent conversion by binding directly to PrP. The enhancement of PrPres formation observed at low concentrations of CR requires further investigation due to the obvious therapeutic implications.

The presence of guanidine in the conversion buffer prevented the inhibition of conversion by CR. This contradicts data that demonstrate inhibition of conversion by CR in the cell-free conversion assay in guanidine buffer (Demaimay et al., 1998, 2000). Reasons for the observed difference may be the source of recombinant PrP, the species of PrP or other experimental conditions. However, the physiological conditions of our conversion assay give more relevance to the assay as a model of conversion and the similarity between our in vitro data and those from cell culture experiments (Rudyk et al., 2000) support the use of more physiologically compatible buffers.

In summary, we have demonstrated that recombinant PrP expressed in a bacterial system and refolded in vitro can be converted into PrPres. In addition, the assay was shown to mimic the in vivo species specificity of transmission of 263K hamster and 87V mouse scrapie between hamster and mouse and will allow the study of TSE transmission barriers. Together, the correlation between the cell-free and the cellular conversion assay in the inhibitory effects of anti-TSE compounds, and the use of a physiologically compatible conversion buffer, make the conversion assay using bacterial recombinant PrP a promising model for discovering and investigating potential prophylactics.


   ACKNOWLEDGEMENTS
 
We would like to acknowledge the Department of Health for funding, Richard Potter for preliminary work on this project, Ian Sylvester for recombinant PrP-expressing clones, Andrew Gill for mass spectrometry, Shalu Patel for circular dichroism spectroscopy and Jean Manson for critical reading of the manuscript.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Basler, K., Oesch, B., Scott, M., Westaway, D., Walchli, M., Groth, D. F., McKinley, M. P., Prusiner, S. B. & Weissmann, C. (1986). Scrapie and cellular PrP isoforms are encoded by the same chromosomal gene. Cell 46, 417–428.[Medline]

Bessen, R. A., Kocisko, D. A., Raymond, G. J., Marsh, R. F., Lansbury, P. T. & Caughey, B. (1995). Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 375, 698–700.[CrossRef][Medline]

Boss, R. P., Koopman, J. P., Theuws, J. l. & Henderson, P. T. (1987). The essential role of the intestinal flora in the toxification of orally-administered benzidine-based dyes: internal exposure of rats to benzidine after intestinal azo reduction. Mut Res 181, 327 (Abstract).

Bossers, A., Belt, P. B. G. M., Raymond, G. J., Caughey, B., de Vries, R. & Smits, M. A. (1997). Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms. Proc Natl Acad Sci U S A 94, 4931–4936.[Abstract/Free Full Text]

Bossers, A., de Vries, R. & Smits, M. A. (2000). Susceptibility of sheep for scrapie as assessed by in vitro conversion of nine naturally occurring variants of PrP. J Virol 74, 1407–1414.[Abstract/Free Full Text]

Caughey, B. & Race, R. E. (1992). Potent inhibition of scrapie-associated PrP accumulation by Congo Red. J Neurochem 59, 768–771.[Medline]

Caughey, B. & Chesebro, B. (1997). Prion protein and the transmissible spongiform encephalopathies. Trends Cell Biol 7, 56–62.[CrossRef]

Caughey, B., Ernst, D. & Race, R. E. (1993). Congo red inhibition of scrapie agent replication. J Virol 67, 6270–6272.[Abstract]

Collins, S. J., Lewis, V. L., Brazier, M., Hill, A. F., Fletcher, A. & Masters, C. L. (2002). Quinacrine does not prolong survival in a murine Creutzfeldt–Jakob disease model. Ann Neurol 52, 503–506.[CrossRef][Medline]

DebBurman, S. K., Raymond, G. J., Caughey, B. & Lindquist, S. (1997). Chaperone-supervised conversion of prion protein to its protease-resistant form. Proc Natl Acad Sci U S A 94, 13938–13943.[Abstract/Free Full Text]

Demaimay, R., Harper, J., Gordon, H., Weaver, D., Chesebro, B. & Caughey, B. (1998). Structural aspects of Congo red as an inhibitor of protease-resistant prion protein formation. J Neurochem 71, 2534–2541.[Medline]

Demaimay, R., Chesebro, B. & Caughey, B. (2000). Inhibition of formation of protease-resistant prion protein by Trypan Blue, Sirius Red and other Congo Red analogs. Arch Virol 16, 277–283.

Doh-Ura, K., Iwaki, T. & Caughey, B. (2000). Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation. J Virol 74, 4894–4897.[Abstract/Free Full Text]

Gilbert, I. H. & Rudyk, H. (1999). Inhibitors of protease-resistant prion formation. Int Antiviral News 7, 78–82.

Griffith, J. S. (1967). Self-replication and scrapie. Nature 215, 1043–1044.[Medline]

Hill, A. F., Antoniou, M. & Collinge, J. (1999). Protease-resistant prion protein produced in vitro lacks detectable infectivity. J Gen Virol 80, 11–14.[Abstract]

Hope, J., Morton, L. J. D., Farquhar, C. F., Multhaup, G., Beyreuther, K. & Kimberlin, R. H. (1986). The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP). EMBO J 5, 2591–2597.[Abstract]

Horiuchi, M. & Caughey, B. (1999). Specific binding of normal prion protein to the scrapie form via a localized domain initiates its conversion to the protease-resistant state. EMBO J 18, 3193–3203.[Abstract/Free Full Text]

Horiuchi, M., Priola, S. A., Chabry, J. & Caughey, B. (2000). Interactions between heterologous forms of prion protein: binding, inhibition of conversion, and species barriers. Proc Natl Acad Sci U S A 97, 5836–5841.[Abstract/Free Full Text]

Ingrosso, L., Ladogana, A. & Pocchiari, M. (1995). Congo red prolongs the incubation period in scrapie-infected hamsters. J Virol 69, 506–508.[Abstract]

Iniguez, V., McKenzie, D., Mirwald, J. & Aiken, J. (2000). Strain-specific propagation of PrPSc properties into baculovirus-expressed hamster PrPC. J Gen Virol 81, 2565–2571.[Abstract/Free Full Text]

Jackson, G. S., Hill, A. F., Joseph, C., Hosszu, L., Power, A., Waltho, J. P., Clarke, A. R. & Collinge, J. (1999). Multiple folding pathways for heterologously expressed human prion protein. Biochim Biophys Acta 1431, 1–13.[Medline]

Jarrett, J. T. & Lansbury, P. T., Jr (1993). Seeding ‘one-dimensional crystallization’ of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell 73, 1055–1058.[Medline]

Kocisko, D. A., Come, J. H., Priola, S. A., Chesebro, B., Raymond, G. J., Lansbury, P. T. & Caughey, B. (1994). Cell-free formation of protease-resistant prion protein. Nature 370, 471–474.[CrossRef][Medline]

Kocisko, D. A., Priola, S. A., Raymond, G. J., Chesebro, B., Lansbury, P. T., Jr & Caughey, B. (1995). Species-specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier. Proc Natl Acad Sci U S A 92, 3923–3927.[Abstract/Free Full Text]

Korth, C., May, B. C. H., Cohen, F. E. & Prusiner, S. B. (2001). Acridine and phenothiazine derivatives as pharmacotherapeutics for prion disease. Proc Natl Acad Sci U S A 98, 9836–9841.[Abstract/Free Full Text]

Locht, C., Chesebro, B., Race, R. & Keith, J. M. (1986). Molecular cloning and complete sequence of prion protein cDNA from mouse brain infected with the scrapie agent. Proc Natl Acad Sci U S A 83, 6372–6376.[Abstract]

McKenzie, D., Bartz, J., Mirwald, J., Olander, D., Marsh, R. & Aiken, J. (1998). Reversibility of scrapie inactivation is enhanced by copper. J Biol Chem 273, 25545–25547.[Abstract/Free Full Text]

Meyer, R. K., McKinley, M. P., Bowman, K. A., Braunfeld, M. B., Barry, R. A. & Prusiner, S. B. (1986). Separation and properties of cellular and scrapie prion proteins. Proc Natl Acad Sci U S A 83, 2310–2314.[Abstract]

Oesch, B., Westaway, D., Walchli, M. & other authors (1985). A cellular gene encodes scrapie PrP27-30 protein. Cell 40, 735–746.[Medline]

Prusiner, S. B. (1991). Molecular biology of prion diseases. Science 252, 1515–1522.[Medline]

Prusiner, S. B., Groth, D. F., McKinley, M. P., Cochran, S. P., Bowman, K. A. & Masiarz, F. R. (1982). Novel infectious pathogens cause brain degeneration in scrapie. Clin Res 30, 531.

Raymond, G. J., Hope, J., Kocisko, D. A. & 12 other authors (1997). Molecular assessment of the potential transmissibilities of BSE and scrapie to humans. Nature 388, 285–288.[CrossRef][Medline]

Rudyk, H., Vasiljevic, S., Hennion, R. M., Birkett, C. R., Hope, J. & Gilbert, I. H. (2000). Screening Congo Red and its analogues for their ability to prevent the formation of PrPres in scrapie-infected cells. J Gen Virol 81, 1155–1164.[Abstract/Free Full Text]

Saborio, G. P., Soto, C., Kascsak, R. J., Levy, E., Kascsak, R., Harris, D. A. & Frangione, B. (1999). Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone. Biochem Biophys Res Commun 258, 470–475.[CrossRef][Medline]

Wells, G. A. H., Scott, A. C., Johnson, C. T., Gunning, R. F., Hancock, R. D., Jeffrey, M., Dawson, M. & Bradley, R. (1987). A novel progressive spongiform encephalopathy in cattle. Vet Rec 121, 419–420.[Medline]

Will, R. G., Ironside, J. W., Zeidler, M. & 7 other authors (1996). A new variant of Creutzfeldt–Jakob disease in the UK. Lancet 347, 921–925.[Medline]

Zhang, F., Zhang, J., Zhou, W., Zhang, B. Y., Hung, T. & Dong, X. P. (2002). Expression of PrPC as HIS-fusion form in a baculovirus system and conversion of expressed PrPsen to PrPres in a cell-free system. Virus Res 87, 145–153.[CrossRef][Medline]

Received 14 October 2002; accepted 3 December 2002.