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Biology

Hsp104 के शोधन, एक प्रोटीन Disaggregase

Published: September 30, 2011
doi:
10.3791/3190
, ,
1Department of Biochemistry and Biophysics,University of Pennsylvania

Summary

यहाँ, हम अत्यधिक सक्रिय Hsp104, hexameric + खमीर, जो जोड़ों एटीपी प्रोटीन disaggregation hydrolysis से एएए प्रोटीन की शुद्धि के लिए एक प्रोटोकॉल का वर्णन. इस योजना के कारनामे His6 टैग से आत्मीयता शुद्धि के लिए का निर्माण<em> ई. कोलाई</em> आयनों मुद्रा क्रोमैटोग्राफी, TEV protease के साथ His6 टैग हटाने, और आकार अपवर्जन क्रोमैटोग्राफी द्वारा पीछा किया.

Abstract

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.

Protocol

1. Hsp104 की अभिव्यक्ति प्लाज्मिड ई. में शुद्धि के लिए नियोजित कोलाई, pPROEX HTB-Hsp104 Hsp104 TRC 26 प्रमोटर के inducible नियंत्रण के तहत खुले पढ़ने फ्रेम शामिल हैं . प्लाज्मिड एन टर्मिनल उनकी 6 टैग कि TEV protease दरा?…

Discussion

समयरेखा: हम अधिक से अधिक Hsp104 गतिविधि के लिए सुझाव है कि पूरे शुद्धि योजना के रूप में तेजी से संभव के रूप में पूरा किया जाना. हालांकि, शुद्धि चरणों की संख्या एक मांग अनुसूची है कि हमेशा व्यावहारिक नहीं ह?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

यह काम (5T32GM008275 22) NIH और एक अमेरिकन हार्ट एसोसिएशन predoctoral फेलोशिप (ईएएस के लिए) से एक अनुदान द्वारा समर्थित किया गया, रसायन विज्ञान जीवविज्ञान (2T32GM071339 06A1) NIH (मेड करने के लिए) से इंटरफ़ेस फैलोशिप, और से अनुदान (1DP2OD002177-01 और NS067354 – 0,110) एनआईएच, एलिसन मेडिकल फाउंडेशन, और बिल और मेलिंडा गेट्स फाउंडेशन (जे एस के लिए).

Materials

Name of the reagent Company Catalogue number
BL21-CodonPlus-RIL Competent Cells Stratagene, Agilent Technologies 230255
2XYT broth USB 75864
Complete, mini, EDTA-free protease inhibitor tablets Roche 1836170
Pepstatin A Sigma P4265
Ni-Sepharose 6 Fast Flow GE Healthcare 17-5318-02
Amicon Ultra-15 centrifugal filter units (MWCO 30,000) Millipore UFC903008
Resource Q – 6ml column GE Healthcare 17-1179-01
proTEV Protease Promega 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
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PurificationHsp104Protein DisaggregaseHexameric AAA+ ProteinATP HydrolysisProtein DisaggregationRenaturationDisordered AggregatesHeat ShockCross-beta Amyloid FibrilsPrionsPhenotypic VariationPreamyloid OligomersE. Coli Orthologue ClpBHsp104 OrthologuesAnimal CellsTherapeutic AgentNeurodegenerative DiseasesMisfolding Of ProteinsToxic Preamyloid OligomersAmyloid Fibrils
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Sweeny, E. A., DeSantis, M. E., Shorter, J. Purification of Hsp104, a Protein Disaggregase. J. Vis. Exp. (55), e3190, doi:10.3791/3190 (2011).
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