Name: purification of hsp104, a protein disaggregase
Uploaded: 2011-09-30T13:00:00
Description: Watch this Scientific Journal Video about Purification of Hsp104, a Protein Disaggregase at JoVE.com

Эта работа была поддержана грантом NIH (5T32GM008275-22) и Американской ассоциации сердца predoctoral стипендий (на EAS); химия-биология интерфейс стипендий из NIH (2T32GM071339-06A1) (МЭР), а также гранты NIH (1DP2OD002177-01 и NS067354-0110), Эллисон Медицинский фонд и Фонд Билла и Мелинды Гейтс (на JS).

1. Выражение Hsp104 <ol><li> Плазмиды используются для очистки в E. палочка, pPROEX-HTB-Hsp104, содержит Hsp104 открытым рамки считывания при индуцируемый контролем промотора TRC 26. Плазмиды производит Hsp104 с N-терминал Его 6-теги, которые могут быть удалены TEV расщепления протеазы. Прео...

Сроки: для максимальной активности Hsp104 мы рекомендуем, чтобы вся схема очистки быть завершены как можно быстрее. Тем не менее, число стадий очистки позволяет напряженный график, что не всегда может быть практичным. Если стадий очистки осуществляется в кратчайшие сроки, время от око?...

<table border="1"><tbody><tr><th> Название реагента </th><th> Компания </th><th> Номер в каталоге </th></tr><tr><td> BL21-CodonPlus-RIL компетентных клеток </td><td> Stratagene, Agilent Technologies </td><td> 230255 </td></tr><tr><td> 2XYT бульон </td><td> USB </td><td> 75864 </td></tr><tr><td> Полная, мини, ЭДТА без таблетки ингибитор протеазы </td><td> Roche </td><td> 1836170 </td></tr><tr><td> Пепстатина </td><td> Сигма </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 Ультра-15 центробежный фильтр единиц (MWCO 30000) </td><td> Millipore </td><td> UFC903008 </td></tr><tr><td> Ресурс Q - 6 мл колонке </td><td> GE Healthcare </td><td> 17-1179-01 </td></tr><tr><td> proTEV протеазы </td><td> Promega </td><td> V6052 </td></tr><tr><td> AcTEV протеазы </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> Анализ образцов </td><td> SPP-400 </td></tr><tr><td> Hsp72 </td><td> Анализ образцов </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

Здесь мы опишем протокол для очистки высокоактивной Hsp104, hexameric AAA + белка из дрожжей, которые пары гидролиза АТФ с белком дезагрегации. Эта схема использует His6 с метками построить для аффинной очистки от<em&gt; Е. палочки</em&gt; Затем анионообменной хроматографии, His6-теги удаления с протеазы ТРВ, и гель-хроматографии.

Очистка Hsp104, белка Disaggregase

Hsp104 is a hexameric AAA+ protein1 from yeast, which couples ATP hydrolysis to protein disaggregation2-10 (Fig. 1). This activity imparts two key ...

Research

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Biology

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Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Автоматизированная гидрофобные взаимодействия хроматографии на колонке выбора для использования в очистки белков

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Chromatographic Purification of Highly Active Yeast Ribosomes

Хроматографической очистки высокоактивной рибосомы дрожжей

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Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Ортогональные очистки белков Ведущий: Малый Биспецифичные тегов Affinity

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Identification of Protein Interacting Partners Using Tandem Affinity Purification

Идентификация белка взаимодействие партнеров, используя тандем аффинной очистки

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Поколение, очистка и характеристика Cell-инвазивных видов белка DISC1

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Очистки белков-Free метод привязки Определение Affinity по Микромасштабные Термофорез

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

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Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...