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://www.jove.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://www.jove.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://www.jove.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://www.jove.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://www.jove.com/files/ftp_upload/53432/53432fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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&amp;nbsp;</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&amp;nbsp;</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...

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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

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

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &#946;-Protein, the Causative Agent of Alzheimer's Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Neuroscience

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (&gt;95%) A&#946;42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the A&#946;40 peptide with identical success and without the need to alter the method.

Herein we report a tailored HPLC purification protocol that yields high-purity amyloid beta 42 (A&#946;42) and amyloid beta 40 (A&#946;40) peptides, capable of oligomer formation. Amyloid beta is a highly aggregation prone, hydrophobic peptide implicated in Alzheimer&#39;s disease. The amyloidogenic nature of the peptide makes its purification a challenge.

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (A&#946;), can present a significant challenge when trying to obtain high ...

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Medicine

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (&#62;98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail.

Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

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 ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease (AD), amyloid-beta peptides form protofibrils in the extracellular space, which act as seeds that gradually grow and mature into large amyloid plaques. Despite this basic understanding, current knowledge of the amyloid fibril structure, composition, and deposition patterns in the brain is limited. One major barrier has been the inability to isolate highly purified amyloid fibrils from brain extracts. Affinity purification and laser capture microdissection-based approaches have been previously used to isolate amyloids but are limited by the small quantity of material that can be recovered. This novel, robust protocol describes the biochemical purification of amyloid plaque cores using sodium dodecyl sulfate (SDS) solubilization with sucrose density gradient ultracentrifugation and ultrasonication and yields highly pure fibrils from AD patients and AD model brain tissues. Mass spectrometry (MS)-based bottom-up proteomic analysis of the purified material represents a robust strategy to identify nearly all the primary protein components of amyloid fibrils. Previous proteomic studies of proteins in the amyloid coronae have revealed an unexpectedly large and functionally diverse collection of proteins. Notably, after refining the purification strategy, the number of co-purifying proteins was reduced by more than 10-fold, indicating the high purity of the isolated SDS insoluble material. Negative staining and immuno-gold electron microscopy allowed confirmation of the purity of these preparations. Further studies are required to understand the spatial and biological attributes that contribute to the deposition of these proteins into amyloid inclusions. Taken together, this analytical strategy is well-positioned to increase the understanding of amyloid biology.

This biochemical purification method with mass spectrometry-based proteomic analysis facilitates the robust characterization of amyloid fibril cores, which may accelerate the identification of targets for preventing Alzheimer's disease.

Proteinaceous fibrillar inclusions are key pathological hallmarks of multiple neurodegenerative diseases. In the early stages of Alzheimer&#39;s disease ...

Isolation of Amyloid Fibrils from Brain Tissue Extract

Source: Upadhyay, A. et al., <a href="https://app.jove.com/v/63816">Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain.</a> J. Vis. Exp. (2022)
This video demonstrates the protocol for isolating&#160; amyloid fibrils from mouse brain tissue extract using density gradient centrifugation and digestion of non-amyloid proteins.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Tissue harvesting and ...

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

Summary

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Selection of Aptamers for Amyloid β-Protein, the Causative Agent of Alzheimer's Disease

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Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Purification of Hsp104, a Protein Disaggregase

Rapid Generation of Amyloid from Native Proteins In vitro

Biochemical Purification and Proteomic Characterization of Amyloid Fibril Cores from the Brain

Isolation of Amyloid Fibrils from Brain Tissue Extract