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Biology

Hsp104纯化，蛋白质Disaggregase

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

Summary

在这里，我们描述一个高度活跃Hsp104，一个六聚AAA +从酵母中的蛋白质，其中夫妇ATP水解蛋白分解净化的协议。该计划利用从亲和纯化His6标签的​​构造<em> E。大肠杆菌</em>其次，His6标签去除TEV蛋白酶，体积排阻色谱阴离子交换层析。

Abstract

Hsp104是一个六聚AAA +蛋白1，从酵母，这夫妇ATP水解蛋白质分解2-10（图1）。这个活动赋予了两个关键的选择性优势。首先，由Hsp104无序聚集复性酵母生存的授权后，各种蛋白质的错误折叠强调，包括热休克3,5,11,12。其次，Hsp104交叉β-淀粉样纤维的重塑使酵母利用无数的朊病 ​​毒（感染性淀粉样）和水库作为一个有益的遗传表型变异13-22。值得注意的是，Hsp104直接重塑preamyloid低聚物和淀粉样纤维，包括酵母朊蛋白Sup35 和 Ure2 23-30组成的。这淀粉样蛋白重塑的功能是专业方面的酵母Hsp104。的大肠杆菌大肠杆菌同源，ClpB，无法改造preamyloid低聚物或淀粉样纤维26,31,32。

Hsp104同源除外，令人困惑，动物王国的生活。事实上，无论是动物细胞中拥有任何酶系统，夫妻蛋白复性（而不是退化）的分类仍然是未知 33-35 。因此，我们和其他人提出，Hsp104可能会被视为一个与特定的蛋白质错误折叠成有毒的preamyloid低聚物和淀粉样纤维 4,7,23,36-38有关的各种神经退行性疾病的治疗剂开发。有没有直接目标与这些疾病有关的汇总物种的治疗。然而，Hsp104溶解毒性低聚物和淀粉样纤维的α-突触核蛋白，这是帕金森氏病23以及39的PrP淀粉样蛋白形式相连组成。更重要的是，Hsp104减少蛋白质聚集和改善帕金森氏病23和亨廷顿氏病38的老鼠模型的神经退行性疾病。理想的情况下，优化治疗和减少副作用，Hsp104将设计和potentiated有选择性地改造的核心问题4,7的疾病的具体聚集。然而，有限的结构和机理的了解如何Hsp104分解聚合结构和不相关的蛋白质的这样一个多样化的剧目挫败这些努力30,40-42。

要了解Hsp104的结构和机制，它是用最少的元件必须研究的纯蛋白质和重组其disaggregase活动。 Hsp104是一个102kDa蛋白与PI〜5.3，ADP或ATP，或在高蛋白质浓度在43-46核苷酸的情况下hexamerizes。在这里，我们描述了一个优化的协议， 从 E的纯化高度活跃，稳定Hsp104 大肠杆菌 。 E.使用大肠杆菌可以简化大规模生产，我们的方法可以进行快速，可靠地为众多Hsp104变种。我们的协议，增加Hsp104的纯度和简化他的6个标签去除，相比以前的提纯方法从E. 大肠杆菌 47。此外，我们的协议是两个较近期的协议 26,48比更轻便和方便。

Protocol

1。对Hsp104的表达质粒在大肠杆菌中聘用为净化大肠杆菌 ，pPROEX HTB – Hsp104，包含的trc启动子的诱导 26控制下Hsp104开放阅读框。质粒产生的N -端他的6个标签，可删除TEV蛋白酶裂解Hsp104。转换成密码子优化E. pPROEX HTB – Hsp104 大肠杆菌 BL21 CodonPlus的RIL细胞（Stratagene公司，安捷伦科技公司）使用一个典型的细菌转化过程（例如按照制造商的指示）…

Discussion

时间轴：最大的Hsp104活动中，我们建议尽可能迅速完成整个净化计 ​​划。然而，纯化步骤，使一个苛刻的时间表，可能并不总是实际的。如果净化步骤是尽快地进行，时间从隔夜表达年底通过孵化2-4小时30 ° C是与TEV蛋白酶约9-11小时。一个潜在的地方，暂停以下TEV裂解步骤。如果绝对必要的，Hsp104可能会被冻结这一步后，上文所述（步骤5.2）的管理单元。解冻后和纯化Hsp104然后必须立即?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

这项工作是支持由来自美国国立卫生研究院（5T32GM008275 22）和美国心脏协会predoctoral的奖学金（EAS）的赠款;化学生物学界面从美国国立卫生研究院（2T32GM071339 06A1）（MED）奖学金;和赠款，从国立卫生研究院（1DP2OD002177 – 01和NS067354 – 0110），埃利森医学基金会，比尔和梅林达盖茨基金会（JS）。

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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Tags

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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