1. Over-expression and purification of recombinant murine wild type and mutant prion proteins in E. coli B. SURIYAMONGKOL 1,2, D. DUGGAN 1,2, V. SEMENCHENKO 3 and D.S. WISHART 1,2, * Departments of 1 Biological Sciences and 2 Computing Science, University of Alberta, Edmonton, Alberta T6G 2E9 3 NanoLife Sciences Group, National Institute for Nanotechnology, Edmonton, Alberta T6G 2M9 * Corresponding author Tel: +1-780-492-0383 Email address: david.wishart@ualberta.ca INTRODUCTION Prion diseases or Transmissible Spongiform Encephalopathies (TSEs) in humans and animals were shown to be caused by structural conversion of cellular prion protein to the infectious scrapie isoform (1). Investigation into these structural changes of both wild type and mutant prion molecules, therefore, rely on high- volume preparations of the prion proteins. In this study, we develop methods to optimize the production and purification of recombinant Syrian Hamster mutant prion proteins in E. coli. GENE CONSTRUCT PREPARATION Figure 1. Protein sequence and expression plasmid map. Residues 90-231 of SHPrP were fused to the His-6 tag of pET-15b for expression and subsequent purification. Protein landmarks are outlined according to information from previous structural studies (2). The visual representation of the construct was created using Plasmapper (3). EXPRESSION LEVELS OF MUTANT PROTEINS Figure 2. Coomassie blue stained gel image of cell lysates of E. coli BL21(DE3) carrying wild type and mutant SHPrPc(90-231) constructs. Pilot expression was performed using a small-volume culture (1 ml). Over-expression of recombinant prion protein was induced by IPTG to a final concentration of 1 mM of the culture. Lysates in lanes 1 to 8 showed intense protein bands (18 kDa) of wild type, P102L, A117V, M129V, D178N, F198S, E200K and M129V/F198S mutant proteins, respectively. PURIFICATION PROTOCOL Figure 3. Nickel-NTA Column Purification. Cells were taken from frozen glycerol stocks and grown in 100 ml LB with Ampicillin selection overnight. This starter culture was used to inoculate 500 ml of M9 media to an O.D. between 0.6 and 1.0. Culture was grown for 1 hour before induction using IPTG. Post induction culture was left to produce SHPrPc for 18 hours. Cells were harvested by centrifugation at 3,000 rpm for 25 minutes at 4ºC, resuspended in lysis buffer (8 M Urea, 10 mM Tris, 100 mM K 2 PO 4, 10 mM reduced Glutathione, pH 8.0) and subjected to 5 rounds of freeze/thaw. Lysate was cleared by centrifugation at 12,000 rpm for 1 hour and 10 minutes. Cleared lysate was added to 35 ml of nickel-NTA resin for sufficient binding time. The column was washed with 100 ml of denaturing buffer (8 M Urea, 10 mM Tris, 100 mM K 2 PO 4, pH 8.0). The proteins were refolded using a 200 ml gradient going from denaturing buffer to refolding buffer (10 mM Tris, 100 mM K 2 PO 4, pH 8.0). The column was subsequently washed with 200 ml of refolding buffer and 100 ml of refolding buffer containing 50 mM Imidazole. Refolded protein was eluted with 200 ml of a competitive nickel-binding buffer (500 mM Imidazole 10 mM Tris, 100 mM K 2 PO 4, pH. 5.6). 5 ml fractions were collected and sampled for eluted prions. Samples were dialysed against appropriate buffers for further concentration. The protocol was adapted from the method described by Zahn et al. (4). PURIFIED PROTEIN SAMPLES Figure 4. Coomassie blue stained gel image of purified wild type SHPrPc(90-231). Small volumes (5-10 µl) of purified protein fractions were analyzed by SDS-PAGE using a 15% acrylamide gel. Protein bands showed an appropriate size of 18 kDa with high purity. The purified samples were subsequently quantified by Bradford assay. OFF-COLUMN REFOLDING For most mutant proteins, we encountered difficulties with on-column refolding and elution under native conditions. Purified mutant proteins were more efficiently eluted from the Ni-NTA column under denaturing conditions, and refolded by dialyzing against appropriate buffers. Yields were decreased due to the last conditioning step. Figure 5. Off-column refolding scheme. Denatured protein solution was added drop- wisely to initial refolding buffer (1.1 GuHCl, 55 mM Tris, 21 mM NaCl, 0.8 mM KCl, 1 mM GSH, 1 mM GSSG, pH 8.2). Total volume of the protein solution was dialyzed against an appropriate buffer using dialysis tubing with 3 kDa cut-off. DETERMINING OPTIMAL BUFFER CONDITION Purified and refolded prion protein was concentrated using an Amicon Ultra centrifugal filter. A small portion of concentrated sample (10-15 µl) was used to perform button test with microdialysis cells to investigate the solubility of the protein in various buffer conditions. The microdialysis cells were incubated in test buffer solutions at room temperature for up to 2 weeks. AVAILABLE PRION PROTEIN SAMPLES Table 1. Summary of the available prion protein samples (sh and mm refer to Syrian Hamster and Mus musculus) CONCLUSION We are continuing to optimize the expression, purification and refolding procedures in order to produce sufficient amounts of each mutant protein to perform complete structural characterization. All constructs are available as BL21(DE3) stab cultures. Purified and refolded protein samples are also available upon request. REFERENCE (1) Prusiner, S.B. (1996). Molecular biology and pathogenesis of prion diseases. Trends Biochem. Sci. 21, 482-487 (2) James, T.L., Liu, H., Ulyanov, N.B. et al. (1997). Solution structure of a 142-residue recombinant prion protein corresponding to the infectious fragment of the scrapie isoform. PNAS 94, 10086-10091 (3) Dong, X., Stothard, P., Forsythe, I.J. and Wishart, D.S. (2004) Plasmapper: a web server for drawing and auto- annotating plasmid maps. Nucleic Acids Res. 1: 32, 660-4. (4) Zahn, R., Liu, A., Lührs, T., Riek, R., et al. (2000) NMR solution structure of the human prion protein. PNAS 97:1, 145-150 ACKNOWLEDGEMENTS We thank our funding sources, PrioNet Canada and Alberta Prion Research Institute and Alberta Ingenuity Fund. Denatured prion protein in 8M Urea or 6M GuHcl 10 volumes of initial refolding buffer with 1.1 M GuHCl Dialyze against 20 volumes of dialysis buffer Recombinant PrPc(90-231) Status Yield (mg/L of culture) Solubility (mg/ml) Buffer Condition sh wild typePurified45450 mM Na 2 PO 4, pH 8.0 sh P102LPurified414.1100 mM K 2 PO 4, pH 8.0 sh A117VPurified21310 mM Tris, 100 mM K 2 PO 4, pH 8.0 sh M129VPurified35310 mM Tris, 100 mM K 2 PO 4, pH 8.0 sh D178NPurified32410-50 mM NaOAc, pH 4.0-5.0 sh F198SPurified35420 mM Tris, 100 mM NaCl, pH 8.0 sh E200KPurified30410-50 mM NaOAc, pH 4.0-5.5 sh M129V /F198S ExpressedIn progress mm wild typeExpressedIn progress 1 2 3 4 5 6 7 8 20 15 kDa 15 20 kDa SHPrPc (90-231) ABSTRACT Prion diseases are a family of fatal neurodegenerative disorders, which affect both humans and animals. In humans, this includes Gerstmann-Straussler-Scheinker (GSS), Fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Familial transmission of these diseases is associated with mutations in the PrP gene. To investigate the molecular basis of these mutations, recombinant Syrian Hamster prion (residues 90-231 - the protease resistant fragment) containing seven different point mutations (P102L, A117V, M129V, D178N, F198S, E200K and M129V/F198S) were over-expressed in E. coli BL21(DE3). The polypeptides were expressed as N-terminal Histidine-tagged fusion proteins and purified using a nickel-NTA agarose resin. Methods for protein refolding, which include on-column refolding and the use of membrane dialysis, were developed according to properties of each of the mutant proteins. Production yields of the target proteins vary from 10 to 45 milligrams per one liter of E. coli culture depending on types of the mutants. Currently, the isotopically labeled samples are being prepared and subsequently characterized by MS and NMR analysis for structural studies. MGSSHHHHHHSSGLVPRGSHMLGQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAV VGGLGGYMLGSAMSRPMMHFGNDWEDRYYRENMNRYPNQVYYRPVDQYNNQNNF VHDCVNITIKQHTVTTTTKGENFTETDIKIMERVVEQMCTTQYQKESQAYYDGRRSS 90 231 Alpha Helices Beta sheets Cysteines involved in disulfide bonds Residues 179 and 214 Fusion tag for purification Sites of Glycosylation. Residues 181 and 197 Growth and expression in M9 media Harvest culture and lyse using freeze/thaw method in denaturing buffer Centrifuge lysate to clear the sample for column loading Load column and wash to clear non-specifically bound protein Use a gradient from denaturing to non-denaturing conditions to slowly refold the prion Elute bound prions Check the concentration of the isolated fractions Dialyse against a choice buffer and store at 4ºC Sample ready for post purification analytical techniques