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

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

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 ...

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

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Bioengineering

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 ...

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

Immunology and Infection

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 ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

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 ...

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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Chapters in this video

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Synthesis of an Intein-mediated Artificial Protein Hydrogel

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

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Purification of Hsp104, a Protein Disaggregase

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Rapid Generation of Amyloid from Native Proteins In vitro