Purification of Hsp104, a Protein Disaggregase

Summary

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.

Abstract

Hsp104 is a hexameric AAA+ protein¹ from yeast, which couples ATP hydrolysis to protein disaggregation^2-10 (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 shock^3,5,11,12. 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 variation^13-22. Remarkably, Hsp104 directly remodels preamyloid oligomers and amyloid fibrils, including those comprised of the yeast prion proteins Sup35 and Ure2^23-30. This amyloid-remodeling functionality is a specialized facet of yeast Hsp104. The E. coli orthologue, ClpB, fails to remodel preamyloid oligomers or amyloid fibrils^26,31,32.

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 unknown^33-35. 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 fibrils^4,7,23,36-38. 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 question^4,7. However, the limited structural and mechanistic understanding of how Hsp104 disaggregates such a diverse repertoire of aggregated structures and unrelated proteins frustrates these endeavors^30,40-42.

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 nucleotide^43-46. 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 protocols^26,48.

Protocol

1. Expression of Hsp104

1. 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. according to manufacturer's instructions). It is important to use a codon-optimized E. coli strain because Hsp104 has an unusual codon bias.

Discussion

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°C with TEV protease is approximately 9-11 hours. One potential place to pause is following the TEV cleavage step. If absolutely nec.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BL21-CodonPlus-RIL Competent Cells	Stratagene, Agilent Technologies	230255
2XYT broth	USB Corp., Affymetrix	75864
Complete, mini, EDTA-free protease inhibitor tablets	Roche Group	1836170
Pepstatin A	Sigma-Aldrich	P4265
Ni-Sepharose 6 Fast Flow	GE Healthcare	17-5318-02
Amicon Ultra-15 centrifugal filter units (MWCO 30,000)	EMD Millipore	UFC903008
Resource Q - 6ml column	GE Healthcare	17-1179-01
proTEV Protease	Promega Corp.	V6052
AcTEV Protease	Invitrogen	12575015
Superose 6 10/300 GL	GE Healthcare	17-5172-01
Hsp40	Assay Designs	SPP-400
Hsp72	Assay Designs	ADI-NSP-555