This work is supported by grants to W.C. from National Institutes of Health Grant AI074809 and The University of Texas M. D. Anderson Cancer Center Institutional Research Grant Program.

1. Prepare MES Buffer
<ol>
	<li>Use MES buffer (0.1 M 2-(N-morpholino)ethanesulfonic acid and 0.9 M NaCl) to prepare both protein-only amyloid as well as stabilized soluble protein oligomers. Weigh 1.95 g of MES and 5.27 g of NaCl. Add H2O and adjust pH to 4.7. Make up the final volume to 100 ml. Store at 4&#186; C for up to 6 months.</li>
</ol>
2. Prepare Protein-only Amyloid Directly from Native Proteins
<ol>
	<li>Weigh protein of choice (any native protein or peptide) and reconstitute to 10 mg/ml in MES buffer.</li>
	<li>Incubate at 65 &#186;C in a waterbath for 4 hr. By the end of the period, white precipitate is visible in solution.</li>
	<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
	<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
	<li>A control sample of native protein can be prepared by following steps 2.1, 2.3, and 2.4 without heat precipitation (step 2.2).</li>
</ol>
3. Prepare Hybrid Amyloid from Stabilized Soluble Oligomers of Native Proteins
<ol>
	<li>Prepare stabilized soluble protein oligomers
	<ol>
		<li>Weigh protein of choice and reconstitute to 10 mg/ml in MES buffer. Leave at room temperature for 15 min.</li>
		<li>Weigh 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and prepare a fresh stock of 20 mg/ml in MES buffer.</li>
		<li>Mix protein and EDC solutions at a ratio of 2:5 (v/v), i.e. 2 mg of protein for every 10 mg of EDC.</li>
		<li>Incubate at room temperature for 2 hr.</li>
		<li>Neutralize the sample with 10% (v/v) of Tris-HCl (1 M, pH 10.5).</li>
		<li>Dialyze the protein in a dialysis cassette overnight to exchange to the desired buffer for further analysis.</li>
		<li>Store the stabilized soluble protein oligomer at 4 &#186;C up to 4 weeks.</li>
	</ol>
	</li>
	<li>Prepare hybrid amyloids from stabilized soluble protein oligomer.
	<ol>
		<li>To prepare DNA-containing amyloid, mix DNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare RNA-containing amyloid, mix RNA and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hr. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
		<li>To prepare heparin-containing amyloid, mix heparin and stabilized soluble protein oligomer at 1:1 ratio (w/w). Leave at 4 &#186;C for at least 2 hrs. During this period, insoluble precipitate is visible in solution when the initial protein concentration is 0.5 mg/ml or higher.</li>
	</ol>
	</li>
</ol>

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We have nothing to disclose.

The method described here offers a rapid and flexible means to prepare amyloid fibrils in vitro from virtually any protein or peptide of choice. Several different types of amyloid can be reliably prepared - protein-only fibrils or hybrid aggregates containing DNA, RNA, &#160;or glycosaminoglycans. The procedures involved are simple, straight forward, and do not require advanced technical training. The amyloid prepared by this method is quite stable and can be safely stored at 4 &#186;C or frozen at -80 &#186;C f...

Proteins are the most abundant biological macromolecules present in all types of cells. They occur in a great variety of sizes, structures, and post-translational modifications, and fulfill an enormous range of important biological functions when in their native forms. More than two dozens of aberrant polypeptides have been implicated in numerous human pathological conditions, such as Alzheimer&#39;s disease, Parkinson disease, &#160;and Type 2 Diabetes1-4. The terminal misfolded proteins accumulate as amyloid fibrils&#160;the insoluble stable aggregates that occur extracellularly or intracellularly.
Despite the implication of specific proteins in certain diseases, increasing evidence supports the notion that all polypeptides have intrinsic properties that enable amyloid transformation. A genome-wide sequence survey identified the &#8220;amylome&#8221;, by which amyloid-prone fragments constitute roughly 15% of all coding peptide segments from E. coli to humans5. Accordingly, an increasing number of functional amyloids, which participate in various important cellular processes, have been discovered in recent years. For example, bacteria assemble amyloids to form biofilm and spore structures that are critical for their survival and pathogenesis6-9. Peptide hormones form amyloid deposits during storage within mammalian secretory granules before being released, further implying that the protein amyloid form can serve beneficial biological functions6,10,11. Moreover, proteins can also assemble into amyloid fibrils to relay critical cellular signals12,13.
For many years, the most studied amyloids are prepared from individual peptides that are associated with human diseases, such as amyloid beta or prion peptide. These peptides usually lack well defined structure and spontaneously form amyloid in solution over time, a process mediated by a partially misfolded intermediate that serves as the precursor of insoluble amyloid. The precursor of amyloid is also called soluble protein oligomer, which is rare and unstable when generated from natural peptides14,15. However, as described earlier, almost any protein in principle can adopt amyloid conformation under the appropriate conditions. We recently established a method to prepare stabilized soluble oligomers of native proteins, which readily form amyloid in the presence of various cofactors16. The resulting amyloid fibrils reflect the complex nature of the terminal protein misfolding aggregates17,18. Here we use the term protein-only amyloid to refer the protein fibrils without cofactors and hybrid amyloid for the fibrils containing nonproteinaceous components.
Previously, various methods have been developed to generate amyloid in vitro by applying conditions known to favor protein misfolding, such as high temperature, hydrophobic environment, and pH variations19-23. However, they associate with various problems, such as low efficiency, reversible folding, large batch variations or slow kinetics. The approach described here is reproducible, easy to scale up, and allows one to precisely control the timing and condition of amyloid conversion.

<table border="0" cellpadding="0" cellspacing="0" width="795">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">2-(N-morpholino)ethanesulfonic acid (MES)</td>
			<td style="width:145px;">Sigma-Aldrich</td>
			<td style="width:164px;">M8250</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">NaCl</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:164px;">BP358-10</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">1-ethyl-3-[3-dimethyl-aminopropyl] carbodiimide hydrochloride (EDC)</td>
			<td style="width:145px;">Thermo/Pierce</td>
			<td style="width:164px;">22980</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">DNA from salmon sperm</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">D1626</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">RNA from torula yeast</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">R6625</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">Heparin from porcine intestinal mucosa</td>
			<td style="width:145px;">Sigma</td>
			<td style="width:164px;">H3149</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:487px;">Tris base</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:164px;">BP152-1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">Equipment:</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:487px;">Material Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:164px;">Catalogue Number</td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:487px;">Water bath</td>
			<td style="width:145px;">Fisher Science</td>
			<td style="width:164px;">Isotemp 210</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;width:487px;">Slide-A-Lyzer dialysis cassette</td>
			<td style="width:145px;">Thermo/Pierce</td>
			<td style="width:164px;">66380</td>
		</tr>
	</tbody>
</table>

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.

The ability to convert native proteins directly to amyloid relies on the conformational change incurred at high temperature in the MES buffer. Precipitation in the solution is a good indication of protein aggregation and possibly amyloid formation. On the other hand, to allow soluble protein oligomer to stabilize, EDC is used to crosslink the proteins to solidify the oligomeric conformation induced in MES buffer. As a result, the stabilized protein oligomers are multimeric proteins (Figure 1), distinct f...

Watch this Scientific Journal Video about Rapid Generation of Amyloid from Native Proteins In vitro at JoVE.com

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.

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 ...

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6.1K vistas.  The University of Texas MD Anderson Cancer Center. 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.