Purification of functional recombinant human mitochondrial Hsp60.

4区 生物学 Q3 Biochemistry, Genetics and Molecular Biology
Methods in enzymology Pub Date : 2024-01-01 Epub Date: 2024-08-31 DOI:10.1016/bs.mie.2024.07.049
Celeste Weiss, Alberto G Berruezo, Shaikhah Seraidy, Avital Parnas, Igor Tascón, Iban Ubarretxena-Belandia, Abdussalam Azem
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引用次数: 0

Abstract

The mitochondrial 60 kDa heat shock protein (mHsp60) is an oligomeric, barrel-like structure that mediates protein folding in cooperation with its cochaperonin Hsp10, in an ATP-dependent manner. In contrast to the extremely stable oligomeric structure of the bacterial chaperonin, GroEL, the human mHsp60 exists in equilibrium between single and double heptameric units, which dissociate easily to inactive monomers under laboratory conditions. Consequently, purification and manipulation of active mHsp60 oligomers is not straightforward. In this manuscript, we present an improved protocol for the purification of functional mHsp60, following its expression in bacteria. This method is based upon a previously published strategy that exploits the notorious instability of mHsp60 to purify the monomeric form, which is subsequently reconstituted to functional oligomers under controlled conditions. In our protocol, we use affinity chromatography on a Ni NTA-agarose resin as the initial step, facilitating purification of substantial amounts of highly pure active protein. The resulting Hsp60 is suitable for both functional and structural analyses, including crystallography and electron cryo-microscopy (cryo-EM) studies, to obtain high resolution structures of the mHsp60 oligomers alone and in various complexes.

纯化功能性重组人线粒体 Hsp60。
线粒体 60 kDa 热休克蛋白(mHsp60)是一种寡聚的桶状结构,它以一种 ATP 依赖性方式与其辅助伴侣蛋白 Hsp10 合作介导蛋白质折叠。与细菌伴侣素 GroEL 极其稳定的低聚物结构不同,人类 mHsp60 存在于单七聚单元和双七聚单元之间的平衡状态,在实验室条件下很容易解离为非活性单体。因此,活性 mHsp60 寡聚体的纯化和操作并不简单。在本手稿中,我们介绍了在细菌中表达 mHsp60 后纯化功能性 mHsp60 的改进方案。这种方法基于之前发表的一种策略,利用 mHsp60 声名狼藉的不稳定性来纯化单体形式,然后在受控条件下将其重组为功能性寡聚体。在我们的方案中,我们使用 Ni NTA-琼脂糖树脂上的亲和层析作为初始步骤,从而促进了大量高纯度活性蛋白质的纯化。得到的 Hsp60 适合进行功能和结构分析,包括晶体学和电子冷冻显微镜(cryo-EM)研究,以获得 mHsp60 寡聚体单独和各种复合物的高分辨率结构。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Methods in enzymology
Methods in enzymology 生物-生化研究方法
CiteScore
2.90
自引率
0.00%
发文量
308
审稿时长
3-6 weeks
期刊介绍: The critically acclaimed laboratory standard for almost 50 years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Each volume is eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with over 500 volumes the series contains much material still relevant today and is truly an essential publication for researchers in all fields of life sciences, including microbiology, biochemistry, cancer research and genetics-just to name a few. Five of the 2013 Nobel Laureates have edited or contributed to volumes of MIE.
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