- Open Access
Green fluorescent protein as a reporter of prion protein folding
- Snezana Vasiljevic†1,
- Junyuan Ren†1,
- YongXiu Yao1,
- Kevin Dalton1,
- Catherine S Adamson1 and
- Ian M Jones1Email author
© Vasiljevic et al; licensee BioMed Central Ltd. 2006
- Received: 28 June 2006
- Accepted: 29 August 2006
- Published: 29 August 2006
The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein.
Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence.
Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.
- Green Fluorescence Protein
- Prion Protein
- Green Fluorescence Protein Fluorescence
- Fluorescence Recovery
- Refold Buffer
The cellular prion protein PrPc is a glycosylinositol phospholipid (GPI) anchored glycoprotein present on neuronal and other cells [1, 2] with a demonstrable ability to bind and transport copper ions [3–6]. The protein is essential for susceptibility to the Transmissible Spongiform Encephalopathies (TSEs) where the accumulation of a disease associated conformational variant, PrPSc, is dependent on the presence of the cellular PrPc isoform (for reviews [7–9]). A role for prion protein in copper metabolism may be linked to cell resistance to oxidative stress and, thereby, to pathology [10–16]. The C-terminal domain of mouse PrPc, whose structure has been determined by NMR, has three α-helices and a short section of antiparallel β-sheet . It folds quickly in vitro to a stable structure largely unaffected by amino acid substitution [18, 19]. By contrast, the N-terminal domain of PrPc is flexibly disordered in the full-length molecule [20, 21]. This region encodes the octarepeat motifs (residues 23–90) responsible for low affinity copper binding [3, 4, 22–24] and the central hydrophobic region of PrPc observed to be toxic to cells in culture , that also binds copper [6, 15, 26] and is involved in the conversion of PrPc to PrPSc [27–29]. Prion diseases have been proposed to be essentially diseases of protein folding [30–32] in which misfolded PrPc, triggered by the presence of PrPSc, forms aggregates associated with toxicity. Equally, misfolded PrPc could be linked to disease through failure to fulfil its normal function, possibly in copper transport [6, 33, 34]. In keeping with these models, antibodies or tagged PrPc that compete for prion protein interaction prevent the accumulation of PrPSc [35, 36] and subsequent pathology [37, 38]. Pathology could also result from aberrant or amplified signalling, leading to apoptosis, a situation mimicked by the binding of antibodies that cross link cell surface PrPc . Interestingly, antibodies that cause apoptosis map to the unstructured domain (residues 95–105) while those binding to the structured C-terminal half of PrPc are not active . Thus, methods that address prion protein folding may help describe the exact link between folding and the various properties ascribed to the PrPc molecule. We have investigated a methodology developed originally to improve the expression of proteins for structural studies [40–42] to report on prion protein folding. Using constructs with endpoints reported previously to alter expression levels  we show that PrPc-GFP fusions protein can be refolded in vitro and that folding is related to the sequence of the PrPc expressed. In addition, mutations that directly affect folding can be selected from a random expression libraries based of the recovery of GFP fluorescence. The use of a co-folding partner thus offers an indirect measure of prion protein folding both in vivo and in vitro.
Establishment of PrPc-GFP refolding in vitro
Use of GFP refolding to assess the role of the extreme N terminus
Use of GFP for direct selection of folding variants
The use of protein fusions as reporters of protein folding and solubility has emerged rapidly and includes use of chloramphenicol acetyltransferase (CAT) , β-galactosidase [60, 61] and secretion by defined bacterial translocation systems . The most well defined system however has been fusion to the N terminus of GFP [40, 42] although fusions within the loops of the folded structure have also been reported . The requirement for increased folding and solubility has been largely driven by the production of proteins for structural studies  but studies with known misfolding proteins such as Alzheimer's amyloid beta peptide have shown that they can be equally applied to the study of folding per se [60, 62]. Here we showed that fusion of GFP to the C-terminus of the mouse prion protein or fragments thereof can provide a measure of the role of prion sequence in folding in vitro and that direct selection of fluorescence in vivo results in PrPc-GFP fusion proteins with altered proprieties of solubility. Refolding of PrPc-GFP fusions was found to be robust and not to result in degradation but marked variation in efficiency was noted when the refolding of individual fragments of PrPc was investigated. In particular, the presence or not of residues 23–28 (KKRPKP), highly conserved in prion sequences , substantially affected refolding in vitro and mirrored their affect on PrPc-GFP fusion protein expression in vivo . The diverse biological properties of this region, including binding of prion protein to charged molecules such as Heparin and GAGs [66–69], suramin  and cellular routing [53, 54] would be consistent with a role on the overall structure of the prion protein. Indeed, restricting movement by N-terminal tethering of PrPc to the cell surface abrogates the only known function of the protein, cellular resistance to oxidative stress . Previous antibody binding studies have suggested that the prion N-terminus may contact the carboxyl domain  and we have previously suggested this interaction may occur between the basic amino terminus and the acidic patch in helix-1 (143DWED146) . Matsunaga et al., using an N-terminally truncated PrPc molecule, previously proposed a model in which the free N-terminal amine of PrPc residue 90 (the truncation point) interacted with the acidic charge cluster in helix-1 following the observation that cryptic epitopes for monoclonal antibody 3F4 within the N-terminus are revealed by titration of acidic residues around Glu 152 . The GFP fluorescence recovery assay described here supports this model but suggests it is residues 23–28 that have a direct role in folding, consistent with binding to the carboxyl domain described elsewhere . While various properties have been ascribed to this short section of charged residues [43, 53, 54, 67, 68, 70, 73] use of refolding in vitro indicates for the first time that these observations could be the result of a role in the overall folding of the molecule.
Prion protein misfolding is thought to underlie its involvement with the TSE diseases and its study, directly or indirectly, may help determine the molecular mechanisms involved. Use of GFP as a folding reporter has been well described but its use as a probe of prion innate folding rather than cellular targeting has not been previously reported. The GFP fluorescence assay we have described may be useful for assessing a number of prion mutations and the interaction of PrPc with its various reported ligands .
E.coli Top 10 (Invitrogen) was used throughout for cloning. Plasmids were transformed into E.coli BL21 DE3 (pLysS) (Novagen) for T7 driven protein production.
Mouse Prnpa allele (accession A23544) and enhanced green fluorescence protein (accession AAC53663) were used throughout. cDNA fragments encoding amino acids 23–231 and the N-terminal residues 23–156, 29–156, 76–156, 23–169 and 29–169 were amplified by the polymerase chain reaction (PCR) to be flanked by restriction sites for Bam H1 and first cloned into baculovirus transfer vector pAcVSVGTMGFP  for expression in insect cells . Each construct was then used as a template to amplify the sequence encoding the fusion of PrPc and eGFP flanking the sequence with restriction sites Nde1 and Xho1 at the 5' at the 3' ends respectively. Fragments were digested with the same enzymes and cloned into pET23a (Novagen) through the same sites to produce PrPc-GFP gene fusions under the control of the T7 promoter.
A degenerate library of prion sequences was created by error prone PCR  and cloned en masse into pET23a upstream of, and in frame with, a sequence encoding eGFP. Several library members were picked at random for nucleotide sequencing to ensure errors had been introduced. The library was maintained in E.coli BL21 pLysS in an un-induced state and induced for fluorescence screening by replica plating to agar containing 2 mM IPTG. Colonies were screened visually after a further 5 hours incubation and positives re-streaked to ensure positivity bred true. Once confirmed, uninduced colonies were re-streaked from the master plate and DNA isolated for sequencing.
Purification of inclusion bodies (IBs)
IBs were prepared by a modified differential solubility regime . Following inoculation of a single colony into Luria broth cultures were induced with IPTG (0.2 mM) at an OD600 of 0.5. Cultures were grown for a further 2 hours and bacteria harvested by centrifugation at 4500 rpm for 20 minutes at 4°C. The pellet was resuspended in 10 ml PBS and the IBs released by sonication on ice for 10 minutes, 1% triton X-100 (v/v) was added to complete solublization and the IBs collected by centrifugation at 4500 rpm for 10 minutes. The pellet was washed repeatedly with 1% Triton X-100 until the purity of the IBs was at least 90 % as judged by SDS-PAGE.
IBs were denatured and reduced at 95°C for 5 minute in 4 M Urea and then clarified by ultracentrifugation. Refolding was initiated by a single 7× dilution step into a buffer containing 50 mM Tris.HCl pH8.5, 1 mM KCl, 2 mM MgCl2. Recovery of fluorescence over time was monitored by periodic fluorescence measurement at 510 nm in a Genios microplate reader (Tecan). Assays were done in duplicate and the average fluorescent units plotted against time. To assess the role of metal ions in refolding buffers were depleted for ions my mixing with chelex-100 (Bio-Rad) as described  and filtering prior to constitution of the assay. Ranging studies showed that the addition of copper above 10 micromolar was found to be generally inhibitory (i.e. inhibited the refolding of GFP only).
Purification of RNA
Total RNA for inclusion in the refolding assay was prepared from SNB cells as described for RNA that stimulates PrPc-PrPSc conversion . Briefly, cells were washed with PBS and resuspended in 1 ml of Trizol (Invitrogen). The lysate was extracted with chloroform and the RNA recovered by precipitation with isopropanol. The pellet was washed with 75% ethanol, air dried, resuspended in RNA-ase free water and quantitated by A260.
Protease K digestion
RNA stimulated partial protection of PrPc was assessed by digestion of the reaction products after refolding with protease K as described .
Protein samples to be analyzed were separated on pre-cast 10% Tris-HCl SDS-polyacrylamide gels (Bio-Rad) and transferred onto Immobilon-P transfer membrane (Millipore). Western blotting was performed as described (Burnette, 1981) except that sensitivity was increased through the use of a biotin conjugated secondary antibody followed by extravidin-peroxidase (Sigma). The membrane was finally developed with BM Chemiluminescence (Roche). The primary antibodies used were prion monoclonal antibodies 6H4 (Prionics) and anti-GFP (Clontech).
We thank Barbara Konig for technical assistance and the UK Medical Research Council and Department for Environment, Food and Rural Affairs (DEFRA) for grant support.
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