This work was supported by a grant from the NIH (5T32GM008275-22) and an American Heart Association predoctoral fellowship (to E.A.S.); a Chemistry-Biology Interface Fellowship from the NIH (2T32GM071339-06A1) (to M.E.D.); and grants from the NIH (1DP2OD002177-01 and NS067354-0110), The Ellison Medical Foundation, and The Bill and Melinda Gates Foundation (to J.S.).

1. Expression of Hsp104
<ol>
<li>The plasmid employed for purification in E. coli, pPROEX-HTb-Hsp104, contains the Hsp104 open-reading frame under the inducible control of the trc promoter26. The plasmid produces Hsp104 with an N-terminal His6-tag that can be removed by TEV protease cleavage. Transform pPROEX-HTb-Hsp104 into codon-optimized E. coli BL21-CodonPlus-RIL cells (Stratagene, Agilent Technologies) using a typical bacterial transformation procedure (e.g. accordi...

No conflicts of interest declared.

Timeline: For maximal Hsp104 activity we recommend that the entire purification scheme be completed as rapidly as possible. However, the number of purification steps makes a demanding schedule that may not always be practical. If the purification steps are carried out as quickly as possible, the time from the end of overnight expression through to the 2-4 hours of incubation at 30&deg;C with TEV protease is approximately 9-11 hours. One potential place to pause is following the TEV cleavage step. If absolutely nec...

<table border="1">
	<tbody>
		<tr>
			<th>Name of the reagent	</th>
			<th>Company	</th>
			<th>Catalogue number</th>
		</tr>
		<tr>
			<td>BL21-CodonPlus-RIL Competent Cells</td>
			<td>Stratagene, Agilent Technologies</td>
			<td>230255</td>
		</tr>
		<tr>
			<td>2XYT broth</td>
			<td>USB	</td>
			<td>75864</td>
		</tr>
		<tr>
			<td>Complete, mini, EDTA-free protease inhibitor tablets</td>
			<td>Roche</td>
			<td>1836170</td>
		</tr>
		<tr>
			<td>Pepstatin A</td>
			<td>Sigma</td>
			<td>P4265</td>
		</tr>
		<tr>
			<td>Ni-Sepharose 6 Fast Flow</td>
			<td>GE Healthcare</td>
			<td>17-5318-02</td>
		</tr>
		<tr>
			<td>Amicon Ultra-15 centrifugal filter units (MWCO 30,000)</td>
			<td>Millipore</td>
			<td>UFC903008</td>
		</tr>
		<tr>
			<td>Resource Q - 6ml column</td>
			<td>GE Healthcare</td>
			<td>17-1179-01</td>
		</tr>
		<tr>
			<td>proTEV Protease</td>
			<td>Promega</td>
			<td>V6052</td>
		</tr>
		<tr>
			<td>AcTEV Protease</td>
			<td>Invitrogen</td>
			<td>12575015</td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL</td>
			<td>GE Healthcare	</td>
			<td>17-5172-01</td>
		</tr>
		<tr>
			<td>Hsp40</td>
			<td>Assay Designs</td>
			<td>SPP-400</td>
		</tr>
		<tr>
			<td>Hsp72</td>
			<td>Assay Designs</td>
			<td>ADI-NSP-555</td>
		</tr>		
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Purification of Hsp104, a Protein Disaggregase

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 ...

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17.2K Görüntüleme.  University of Pennsylvania. Burada, son derece aktif Hsp104 hexameric AAA + protein mayası, protein ayrıştırılmasıyla çiftler ATP hidrolizi arıtma için bir protokol açıklar. Bu düzeni yakınlık arıtma için His6 etiketli inşa patlatır E. coli Anyon-değiştirici kromatografi, TEV proteaz His6-tag kaldırılması, ve boyut dışlama kromatografisi izledi.