The authors would like to acknowledge Natalie Doude and David Westerway (University of Alberta) for providing us Sho cDNA, Christophe Chevalier (INRA) for plasmid construction, Michel Br&#233;mont (INRA) for providing us BL21Sol bacteria; Christine Longin (INRA) for TEM assistance and Edith Pajot-Augy (INRA) for comments and proofreading.

1. Generation of Plasmid and Shadoo Expression
Note: The gene encoding murine Shadoo protein (Shadoo25-122), was subcloned into the expression vector pET-28 11. This clone was transformed into E. coli SoluBL21 bacterial strain, to express Shadoo protein with a N-terminal hexahistidine tag, (HHHHHHHHHHSSGHIDDDDKHMKGGRGGARGSARGVRGGARGASRVRVRP APRYGSSLRVAAAGAAAGAAAGVAAGLATGSGWRRTSGPGELGLEDDENGAMGGNGTDRGVYSYWAWTSG) as explained previously 11. The plasmid can...

<ol>
	<li>Biasini, E., Turnbaugh, J. A., Unterberger, U., Harris, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+protein+at+the+crossroads+of+physiology+and+disease.">Prion protein at the crossroads of physiology and disease.</a> Trends in Neurosciences. 35, 92-103 (2012).</li><li>Colby, D. W., Prusiner, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=De+novo+generation+of+prion+strains.">De novo generation of prion strains.</a> Nature Reviews. Microbiology. 9, 771-777 (2011).</li><li>Chiti, F., Dobson, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding,+functional+amyloid,+and+human+disease.">Protein misfolding, functional amyloid, and human disease.</a> Annual Review of Biochemistry. 75, 333-366 (2006).</li><li>Nelson, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+the+cross-beta+spine+of+amyloid-like+fibrils.">Structure of the cross-beta spine of amyloid-like fibrils.</a> Nature. 435, 773-778 (2005).</li><li>Murphy, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+amyloid+formation+and+membrane+interaction+with+amyloidogenic+proteins.">Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins.</a> Biochimica et Biophysica Acta. 1768, 1923-1934 (2007).</li><li>Steunou, S., Chich, J. F., Rezaei, H., Vidic, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosensing+of+lipid-prion+interactions:+insights+on+charge+effect,+Cu(II)-ions+binding+and+prion+oligomerization.">Biosensing of lipid-prion interactions: insights on charge effect, Cu(II)-ions binding and prion oligomerization.</a> Biosensors &amp; Bioelectronics. 26, 1399-1406 (2010).</li><li>Watts, J. C., Westaway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prion+protein+family:+diversity,+rivalry,+and+dysfunction.">The prion protein family: diversity, rivalry, and dysfunction.</a> Biochimica et Biophysica Acta. 1772, 654-672 (2007).</li><li>Westaway, D., Daude, N., Wohlgemuth, S., Harrison, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PrP-like+proteins+Shadoo+and+Doppel.">The PrP-like proteins Shadoo and Doppel.</a> Topics in Current Chemistry. 305, 225-256 (2011).</li><li>Baillod, P., Garrec, J., Tavernelli, I., Rothlisberger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+versus+doppel+protein+misfolding:+new+insights+from+replica-exchange+molecular+dynamics+simulations.">Prion versus doppel protein misfolding: new insights from replica-exchange molecular dynamics simulations.</a> Biochemistry. 52, 8518-8526 (2013).</li><li>Daude, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+Shadoo+proteins+convert+to+amyloid-like+forms+under+native+conditions.">Wild-type Shadoo proteins convert to amyloid-like forms under native conditions.</a> Journal of Neurochemistry. 113, 92-104 (2010).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shadoo+binds+lipid+membranes+and+undergoes+aggregation+and+fibrillization.">Shadoo binds lipid membranes and undergoes aggregation and fibrillization.</a> Biochemical and Biophysical Research Communications. 438, 519-525 (2013).</li><li>Watts, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CNS+glycoprotein+Shadoo+has+PrP(C)-like+protective+properties+and+displays+reduced+levels+in+prion+infections.">The CNS glycoprotein Shadoo has PrP(C)-like protective properties and displays reduced levels in prion infections.</a> The EMBO Journal. 26, 4038-4050 (2007).</li><li>Watts, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-resistant+prions+selectively+decrease+Shadoo+protein.">Protease-resistant prions selectively decrease Shadoo protein.</a> PLoS Pathogens. 7, e1002382 (2011).</li><li>Westaway, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Down-regulation+of+Shadoo+in+prion+infections+traces+a+pre-clinical+event+inversely+related+to+PrP(Sc)+accumulation.">Down-regulation of Shadoo in prion infections traces a pre-clinical event inversely related to PrP(Sc) accumulation.</a> PLoS Pathogens. 7, e1002391 (2011).</li><li>Chevalier, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PB1-F2+influenza+A+virus+protein+adopts+a+beta-sheet+conformation+and+forms+amyloid+fibers+in+membrane+environments.">PB1-F2 influenza A virus protein adopts a beta-sheet conformation and forms amyloid fibers in membrane environments.</a> The Journal of Biological Chemistry. 285, 13233-13243 (2010).</li><li>Tarus, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomerization+paths+of+the+nucleoprotein+of+influenza+A+virus.">Oligomerization paths of the nucleoprotein of influenza A virus.</a> Biochimie. 94, 776-785 (2012).</li><li>Biancalana, M., Koide, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+of+Thioflavin-T+binding+to+amyloid+fibrils.">Molecular mechanism of Thioflavin-T binding to amyloid fibrils.</a> Biochimica et Biophysica Acta. 1804, 1405-1412 (2010).</li><li>Minic, J., Sautel, M., Salesse, R., Pajot-Augy, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+system+as+a+screening+tool+for+pharmacological+assessment+of+g+protein+coupled+receptors.">Yeast system as a screening tool for pharmacological assessment of g protein coupled receptors.</a> Current Medicinal Chemistry. 12, 961-969 (2005).</li><li>Schein, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cool+way+to+make+proteins.">A cool way to make proteins.</a> Nature Biotechnology. 22, 826-827 (2004).</li><li>Sorensen, H. P., Mortensen, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+genetic+strategies+for+recombinant+protein+expression+in+Escherichia+coli.">Advanced genetic strategies for recombinant protein expression in Escherichia coli.</a> Journal of Biotechnology. 115, 113-128 (2005).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disruption+of+glycosylation+enhances+ubiquitin-mediated+proteasomal+degradation+of+Shadoo+in+Scrapie-infected+rodents+and+cultured+cells.">Disruption of glycosylation enhances ubiquitin-mediated proteasomal degradation of Shadoo in Scrapie-infected rodents and cultured cells.</a> Molecular Neurobiology. 49, 1373-1384 (2014).</li><li>Englund, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+and+biosynthesis+of+glycosyl+phosphatidylinositol+protein+anchors.">The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors.</a> Annual Review of Biochemistry. 62, 121-138 (1993).</li><li>Puig, B., Altmeppen, H., Glatzel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+GPI-anchoring+of+PrP:+implications+in+sorting+and+pathogenesis.">The GPI-anchoring of PrP: implications in sorting and pathogenesis.</a> Prion. 8, 11-18 (2014).</li></ol>

The authors have nothing to disclose.

An easy and efficient protocol for both expression and purification of a large amount of recombinant mouse Shadoo protein is presented. The method described allows successful solubilization and purification of (His6)-tagged recombinant Shadoo protein. It is important, as pointed out in the title, that when expressed in a prokaryotic bacterium E. coli Shadoo accumulates in inclusion bodies. Prior to Shadoo purification, bacteria have to be lysed by sonication in a buffer containing Triton X-100. Subseq...

Prion neurodegenerative diseases, which include bovine spongiform encephalopathy in cattle, scrapie in sheep and Creutzfeldt-Jakob disease in humans, are fatal and incurable. Prion diseases are characterized by conformational changes of cellular prion protein mainly composed of &#945;-helices, into the cross-&#946;-sheet enriched amyloid conformer 1,2. Cross-&#946;-structures contain densely packed and highly ordered crystalline-like &#946;-sheets stabilized with hydrogen bonds 3,4. Prion amyloids can self-replicate due to the &#946;-strands at the growing edge that provide a template for recruiting and converting a monomeric protein unit.
According to the &#34;protein only&#34; hypothesis, the amyloid conformer of prion protein is the sole infectious agent. However, some other biomolecules have been proposed to be essential to prion disorders. For instance, cell membranes are thought to be a place where conversion takes place and some negatively charged lipids have been shown to enhance the conversion process 5,6. In addition, some proteins may also be involved in prion pathologies. Specifically, there are two other members of the prion protein family: Doppel and Shadoo 7,8. Doppel has a relatively rigid structure stabilized with di-sulfide bridges and fails to self-polymerize and aggregate to amyloid fibrils 9. In contrast, similar to prion protein, Shadoo can adopt a &#946;-sheet-enriched structure and self-associate into amyloid fibril structures. It was shown that Shadoo can fibrillate under native conditions or upon binding negatively charged membranes 10,11. Conversion of Shadoo into amyloid-like fibers may be associated with pathologies. Indeed, the mechanisms by which amyloid-like structures cause disease are poorly understood.
Shadoo bears a hydrophobic domain (HD) and a series of tandem Arg/Gly repeats similar to the N-terminal part of prion protein (Figure 1A). As the N-terminal part of prion protein, Shadoo is highly positively charged and appears to be a natively unstructured protein 11,12. Shadoo seems to be functionally related to prion disorders since it can directly bind prion protein. In addition, its expression is down regulated during prion pathology 13,14. However, the role of Shadoo in prion disease is not yet established.
We developed a plasmid carrying the coding sequence of the mouse Shadoo gene. The plasmid was used to transform E. coli for production of N-terminal His6 fusion Shadoo protein. This expression system is well established in our laboratory and is commonly used in our ongoing projects 6,15,16. An important issue for expression of Shadoo is the choice of the E. coli strain. While competent BL21 bacterial strain is commonly used for expression of recombinant proteins Shadoo was successfully expressed and purified only from the transformed SoluBL21 bacterial strain. SoluBL21 competent E. coli is an improved mutant of BL21 host strain developed for the production of proteins whose expression in the parent BL21 gave no detectable soluble product. High-level expression of Shadoo in SoluBL21 E. coli leads to accumulation of the protein in inclusion bodies. As a general feature, when a non-native protein is highly expressed in E. coli, this protein tends to accumulate in insoluble inclusion bodies. Shadoo probably aggregates through non-covalent hydrophobic or ionic interactions (or a combination of both) to highly enriched dynamic structures formed by the protein folded to various degrees. In consequence, the purification comprises at least two steps: (i) a selective separation of the protein from other biomolecules in a denaturation medium, and (ii) a renaturation of the purified protein using in vitro folding techniques.
The selective separation of Shadoo was achieved in a buffer solution containing 8M urea (or alternatively 6M Guanidine-HCl). The urea removal and the renaturation of the protein can be done by applying various protocols: (i) renaturation in an acidic pH solution to obtain an unstructured monomeric Shadoo protein, or (ii) renaturation in a solution of pH &#8805; 7 to obtain Shadoo polymerized into stable amyloid fibers with characteristic cross-&#946;-sheet motifs.

<table>
	<tbody>
		<tr>
			<td>AKTA FPLC&#160;</td>
			<td>GE, Amersham</td>
			<td># 1363</td>
			<td></td>
		</tr>
		<tr>
			<td>HiLoad 16/60 Superdex</td>
			<td>GE Healthcare</td>
			<td>17-1069-01</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTrap IMAC</td>
			<td>GE Healthcare</td>
			<td>GE17-0920-05</td>
			<td></td>
		</tr>
		<tr>
			<td>HiPrep26/10 Desalting column</td>
			<td>GE Healthcare</td>
			<td>GE17-5087-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SoluBL21 Competent E. coli</td>
			<td>Genlantis</td>
			<td>C700200</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-28b+</td>
			<td>Novogen</td>
			<td>69865-3</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758</td>
			<td></td>
		</tr>
		<tr>
			<td>LB-Broth medium</td>
			<td>Sigma-Aldrich</td>
			<td>L3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Biophotometar</td>
			<td>Eppendorf</td>
			<td>550507804</td>
			<td></td>
		</tr>
		<tr>
			<td>Trito X-100</td>
			<td>Life Technologies</td>
			<td>85111</td>
			<td></td>
		</tr>
		<tr>
			<td>NiSO4</td>
			<td>Sigma-Aldrich</td>
			<td>656895</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>RES3098T</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazol</td>
			<td>Sigma-Aldrich</td>
			<td>I5513</td>
			<td></td>
		</tr>
		<tr>
			<td>Gdn-HCl</td>
			<td>Sigma-Aldrich</td>
			<td>G4505</td>
			<td></td>
		</tr>
		<tr>
			<td>protein size marker&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>26620</td>
			<td></td>
		</tr>
	</tbody>
</table>

purification and refolding to amyloid fibrils of (his)6-tagged recombinant shadoo protein expressed as inclusion bodies in e. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni2+-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

About 5-10 mg of purified Shadoo protein is obtained per liter of bacterial culture. A two-step purification is needed to obtain recombinant Shadoo of high purity. The first step is performed by affinity chromatography with a Ni2+-charged column that retains His-tagged-proteins. Eluted fractions are subjected to SDS-PAGE and stained with Coomassie Brilliant Blue (Figure 5A). Fractions containing Shadoo protein are pooled and further purified by size-exclusion chromatography which separates pro...

Watch this Scientific Journal Video about Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli at JoVE.com

Purification and Refolding to Amyloid Fibrils of His6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein ...

purification-refolding-to-amyloid-fibrils-his6-tagged-recombinant

Research

JoVE Journal

Biology

14.0K Görüntüleme.  INRA. A two-step chromatographic method is described for the purification of recombinant Shadoo protein expressed as inclusion bodies in Escherichia coli, as well as a protocol to fibrillate purified Shadoo into amyloid structures. 