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 be requested from the authors. The molecular weight of Shadoo calculated from its amino acid sequence is 12,243.2 Da.
Note: Perform all manipulations with bacteria under a laminal flow hood or close to Bunsen flame.
<ol>
	<li>Transform competent SoluBL21 bacteria with the pET-28-His6-Shadoo plasmid, using a standard heat shock protocol. For this mix 1 to 5 &#181;l of the plasmid (typically 10 pg to 100 ng) into 50 &#181;l of the bacteria in a tube. Perform the heat shock at 42 &#176;C for 45 sec.</li>
	<li>Culture transformed bacteria at 37 &#176;C until A600 of 0.5-0.7 in Luria-Bertani medium supplemented with 40 mg/ml kanamycin (Figure 1B).</li>
	<li>Induce Shadoo expression O/N by adding 240 &#181;g/ml isopropyl-&#946;-D-thiogalactopyranoside, ITPG, to the culture. Culture bacteria under shaking at 150 rpm at 37 &#176;C. Note: Typically, bacterial culture reaches A600 of 3 O/N. 
		Note: Transformed bacteria can be stored at -80 &#176;C for future protein production. For this, add 10%-50% (v/v) sterile glycerol into bacterial culture aliquots and freeze them at -80 &#176;C.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53432/53432fig1.jpg" /> 
	Figure 1. Scheme of Shadoo expression construct, bacteria transformation and induction of Shadoo expression (as described in step 1). (A) The expression construct for Shadoo encodes a hexahistidine tag fused to the N-terminus of the protein, Nt. The scheme shows that Shadoo protein contains basic arginine/glycine repeats (green cylinder), a hydrophobic domain (HD), and a single N-glycosylation site (CHO), followed by a C-terminus (Ct). (B) pET-28-His6-Shadoo plasmid is transformed into SoluBL21 bacteria by heat shock for 45 sec at 42 &#176;C. Transformed bacteria are plated on selective agar containing 40 &#181;g/ml kanamycin. A single colony from the plate is used to inoculate a culture in a 1,000-3,000 ml shaker flask. Expression of the recombinant protein is induced with 1 mM IPTG. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53432/53432fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Purification of Shadoo Protein by Low Pressure Liquid Chromatography (LPLC)
<ol>
	<li>Cell lysis and Inclusion bodies preparation
		<ol>
			<li>Collect the bacterial suspension and transfer it into centrifugation tubes (Figure 2).</li>
			<li>Spin at 2,500 x g, for 20 min at 4 &#176;C. Remove supernatant by decanting and resuspend pellets in 15 ml buffer (50 mM Tris, 10 mM EDTA, pH 8 with anti-protease cocktail) for each pellet derived from 500 ml of cell culture. 
				Note: Resuspended pellets can be frozen and stored at -20 &#176;C for several days. Freezing facilitates bacterial cells lysis.</li>
			<li>Check the over-expression of recombinant protein using crude bacteria pellets by SDS-PAGE analysis and Coomassie staining.
				<ol>
					<li>For this, centrifuge 1 ml of bacterial culture to pellet cells. Add 100 &#181;l Laemmli denaturation solution (277.8 mM Tris-HCl, pH 6.8, 4.4% SDS, 44.4% w/v glycerol, 0.02% bromophenol blue) to pellet and heat at 100 &#176;C for 5 min.</li>
					<li>Load 10 &#181;l of the sample to a 12% acrylamide gel, run SDS-PAGE and visualize separated proteins by Coomassie staining.</li>
				</ol>
			</li>
			<li>Add Triton X-100 to 0.5% final concentration to the completely resuspended pellet from 2.1.2).</li>
			<li>Incubate the suspension in water bath at 37 &#176;C for 30 min.</li>
			<li>Sonicate the suspension for 5 min at 6-12 microns peak-to-peak amplitude in ice-cold water to lyse bacterial cells.</li>
			<li>Transfer sonicated suspension into clean 50 ml centrifugation tubes.</li>
			<li>Centrifuge tubes at 15,000 x g for 15 min at 4 &#176;C.</li>
			<li>Remove supernatant by decanting and add 5 ml of the binding buffer (20 mM Tris-HCl buffer, pH 7.4, 0.5 M NaCl, 8 M Urea, 5 mM Imidazole) per tube.</li>
			<li>Place tubes on the rotating wheel O/N to completely resuspend proteins from inclusion bodies.</li>
		</ol>
	</li>
</ol>
<img alt="Figure 2" src="/files/ftp_upload/53432/53432fig2.jpg" /> 
	Figure 2. Scheme of purification of inclusion bodies (as described in step 2.1). Transformed bacteria expressing Shadoo are harvested by centrifugation and resuspended in the lysis buffer. The suspension is incubated at 37 &#176;C for 30 min, and then sonicated to mechanically break bacterial cells. Sonication is done on ice to prevent sample heating. Broken cells are harvested by centrifugation and resuspended in a buffer containing 8 M urea to solubilize proteins from inclusion bodies. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53432/53432fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Ni 2+-affinity chromatography 
		Note: Chromatography is performed on FPLC System using a Ni2+-charged, 5 ml Sepharose chelating column (Figure 3A). All solutions are loaded on the column with a flow rate of 1 ml/min.
		<ol>
			<li>Inject 10 ml of 0.2 M NiSO4 water solution into the Sepharose column in order to charge it with Ni2+-ions.</li>
			<li>Equilibrate the column charged with Ni2+-ions by injecting 30-50 ml of the binding buffer containing a low concentration of imidazole (20 mM Tris-HCl buffer, pH 7.4, 0.5 M NaCl, 8 M Urea, 5 mM Imidazole).</li>
			<li>Load the urea-solubilized proteins with a 50 ml injection loop.</li>
			<li>Recover the flow-through fraction by thoroughly washing the column with 10 ml low imidazole binding buffer (20 mM Tris-HCl buffer, pH 7.4, 0.5 M NaCl, 8 M Urea, 5 mM Imidazole) in order to remove a maximum of unbound proteins from the column.</li>
			<li>Wash the column with 50-100 ml of the washing buffer (20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 8 M urea, 80 mM imidazole) to elute proteins non-specifically bound to the column.</li>
			<li>Apply a 15 min linear gradient of 80-800 mM Imidazole in the buffer containing 20 mM Tris-HCl, pH 7.4, 0.5 M NaCl, 8 M Urea. Collect 1 ml fractions during the gradient application. Note: His-tagged-proteins are eluted with a gradient of imidazole-containing buffer (used as a competitor).</li>
			<li>Take a 8 &#181;l aliquot of each fraction and add 4 &#181;l 4x Laemmli denaturation solution (see 2.1.3.1). Heat solutions at 100 &#176;C for 5 min.</li>
			<li>Load 10 &#181;l of protein size marker and 10 &#181;l of each sample to the 12% acrylamide gel, run SDS-PAGE and visualize separated proteins by Coomassie staining. Note: The molecular weight of Shadoo calculated form its amino acids sequence is 12,243.2 Da.</li>
			<li>Pool all fractions containing Shadoo.</li>
		</ol>
	</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53432/53432fig3.jpg" /> 
	Figure 3. Purification procedure (as described in steps 2.2-2.4). (A) Ni2+-ions can be coordinated by histidine residues, which allows the selective purification of polyhistidine-tagged proteins. Imidazole is used to elute the protein, through its ability to coordinate with the Ni2+-ion and displace the bound protein. (B) Proteins of varying hydrodynamic radius that were retained on the Ni2+-column are separated onto a size exclusion column. Proteins are eluted from the largest to the smallest ones. (C) Prior to use, fractions containing Shadoo are pooled and desalted to remove urea, imidazole and salts. Quality control assays include SDS-PAGE/Coomassie staining, Western blotting. Purified Shadoo can be freeze-dried. Lyophilized Shadoo is stable for months when stored at -20 &#176;C. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53432/53432fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Size exclusion chromatography 
		Note: A second purification step consists in the size exclusion chromatography which separates Shadoo protein from the other proteins co-eluted during the first purification step (Figure 3B).
		<ol>
			<li>Equilibrate Superdex column of 120 ml total bed volume by injecting 250 ml of 20 mM Tris-HCl buffer, pH 7.4, 0.5 M NaCl, 8 M Urea at the flow rate of 1.5 ml/min.</li>
			<li>Load pooled fractions containing Shadoo from 2.2.9 on the equilibrated column. Collect 1 ml fractions after the column-void volume of 40 ml upon the constant flow of the above buffer.</li>
			<li>Take a 10 &#181;l aliquot of each fraction and perform a SDS-PAGE analysis and Coomassie staining as described above to find out which fractions contain Shadoo.</li>
			<li>Pool fractions containing Shadoo.</li>
		</ol>
	</li>
	<li>Desalting 
		Note: Desalting procedure removes salts, imidazole and urea to obtain recombinant Shadoo protein in a buffer suitable for further biophysical and biochemical studies (Figure 3C). The same result can be obtained by dialyzing samples against the buffer.
		<ol>
			<li>Equilibrate a desalting column of 53 ml with 150 ml of 10 mM ammonium acetate buffer, pH 5, at flow rate of 5 ml/min.</li>
			<li>Load pooled Shadoo fractions on the column. Upon injection of the ammonium acetate buffer collect manually fractions that show an increase of the signal at 280 nm at the output of the UV detector.</li>
			<li>Freeze-dry the desalted Shadoo and store it at -20 &#176;C.</li>
			<li>Dissolve lyophilized Shadoo in an adequate buffer, such as 10 mM sodium acetate buffer, pH 5, prior to use. 
				Note: The polyhistidine tag can be removed with enterokinase.</li>
		</ol>
	</li>
</ol>
3. In Vitro Assembling of Shadoo into Amyloid Fibrils at Physiological pH
Note: The Shadoo protein aggregates and spontaneously forms fibrils at pH &#8805; 7 11.
<img alt="Figure 4" src="/files/ftp_upload/53432/53432fig4.jpg" /> 
	Figure 4. Scheme of Shadoo refolding to amyloid fibrils (as described in step 3). Purified Shadoo spontaneously converts to amyloid fibrils when dissolved in buffer of pH &#8805; 7. In an acidic solution, Shadoo converts to amyloid fibrils upon incubation with 1 M Gdn-HCl while shaking. Quality control assays include electronic microscopy and ThT staining. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53432/53432fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Dilute Shadoo to a final concentration of 2 &#181;M in a 20 mM Tris-HCl buffer, pH 7.4. Measure the concentration by measuring the optical density of the solution at 280 nm and using the extinction coefficient deduced from Shadoo amino acid composition of 20,970.</li>
	<li>Incubate 500 &#181;l of the solution in a conical plastic tube at 4 &#176;C for a couple of weeks, to allow spontaneous protein conversion to amyloid structures (Figure 4).</li>
	<li>Add freshly prepared thioflavin T (ThT) to a final concentration of 10 &#181;M to a 100 &#181;l aliquot of the Shadoo solution. Incubate the solution for 5 min at RT. Measure fluorescence emission from 460 to 520 nm using an excitation wavelength of 435 nm to verify the presence of amyloid structures 17.</li>
</ol>
4. In Vitro Assembling of Shadoo to Amyloid Fibrils at Acidic pHs
<ol>
	<li>Dilute Shadoo in 1 M GdnHCl, 20 mM Na-acetate buffer, pH 5 to a final concentration of 2 &#181;M.</li>
	<li>Incubate 500 &#181;l of this solution in a conical tube with continuous shaking at 37 &#176;C, for 3-7 days.</li>
	<li>Check the presence of amyloid fibrils by adding ThT to an aliquot of the solution as in 3.3.</li>
	<li>Dialyze obtained suspension against 10 mM Na-acetate buffer, pH 5.0, to purify Shadoo amyloid fibrils.</li>
	<li>Store purified fibrils at +4 &#176;C.</li>
</ol>
<img alt="Figure 5" src="/files/ftp_upload/53432/53432fig5.jpg" /> 
	Figure 5. Representative results. Protein fractions eluted from Ni2+-charged chelating column (A) and size exclusion column (B) were analyzed using SDS-PAGE and Coomassie staining. Identification of purified Shadoo was assessed using SPRN anti-Shadoo antibody (C). <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<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-his-6-tagged-recombinant

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

14.1K Views.  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. 

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

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Purification of Hsp104, a Protein Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key selective advantages. First, renaturation of disordered aggregates by Hsp104 empowers yeast survival after various protein-misfolding stresses, including heat shock3,5,11,12. Second, remodeling of cross-beta amyloid fibrils by Hsp104 enables yeast to exploit myriad prions (infectious amyloids) as a reservoir of beneficial and heritable phenotypic variation13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure223-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils26,31,32.
Hsp104 orthologues are found in all kingdoms of life except, perplexingly, animals. Indeed, whether animal cells possess any enzymatic system that couples protein disaggregation to renaturation (rather than degradation) remains unknown33-35. Thus, we and others have proposed that Hsp104 might be developed as a therapeutic agent for various neurodegenerative diseases connected with the misfolding of specific proteins into toxic preamyloid oligomers and amyloid fibrils4,7,23,36-38. There are no treatments that directly target the aggregated species associated with these diseases. Yet, Hsp104 dissolves toxic oligomers and amyloid fibrils composed of alpha-synuclein, which are connected with Parkinson's Disease23 as well as amyloid forms of PrP39. Importantly, Hsp104 reduces protein aggregation and ameliorates neurodegeneration in rodent models of Parkinson's Disease23 and Huntington's disease38. Ideally, to optimize therapy and minimize side effects, Hsp104 would be engineered and potentiated to selectively remodel specific aggregates central to the disease in question4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors30,40-42.
To understand the structure and mechanism of Hsp104, it is essential to study the pure protein and reconstitute its disaggregase activity with minimal components. Hsp104 is a 102kDa protein with a pI of ~5.3, which hexamerizes in the presence of ADP or ATP, or at high protein concentrations in the absence of nucleotide43-46. Here, we describe an optimized protocol for the purification of highly active, stable Hsp104 from E. coli. The use of E. coli allows simplified large-scale production and our method can be performed quickly and reliably for numerous Hsp104 variants. Our protocol increases Hsp104 purity and simplifies His6-tag removal compared to a previous purification method from E. coli47. Moreover, our protocol is more facile and convenient than two more recent protocols26,48.

Here, we describe a protocol for the purification of highly active Hsp104, a hexameric AAA+ protein from yeast, which couples ATP hydrolysis to protein disaggregation. This scheme exploits a His6-tagged construct for affinity purification from E. coli followed by anion-exchange chromatography, His6-tag removal with TEV protease, and size-exclusion chromatography.

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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

Summary

Explore More Videos

Chapters in this video

Patch Clamp Recording of Ion Channels Expressed in Xenopus Oocytes

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamAΔ41

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Purification of Hsp104, a Protein Disaggregase

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli