来自……的重组Skd3（人ClpB）蛋白和烟草蚀纹病毒（TEV）蛋白酶的表达与纯化
Expression and Purification of Recombinant Skd3 (Human ClpB) Protein and Tobacco Etch Virus (TEV) Protease from .
作者信息
Cupo Ryan R, Shorter James
机构信息
Department of Biochemistry and Biophysics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
Pharmacology Graduate Group, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA.
出版信息
Bio Protoc. 2020 Dec 5;10(23):e3858. doi: 10.21769/BioProtoc.3858.
Abstract
Skd3 (encoded by human ) is a mitochondrial AAA+ protein comprised of an N-terminal ankyrin-repeat domain and a C-terminal HCLR-clade nucleotide-binding domain. The function of Skd3 has long remained unknown due to challenges in purifying the protein to high quality and near homogeneity. Recently we described Skd3 as a human mitochondrial protein disaggregase that solubilizes proteins in the mitochondrial intermembrane space. This protocol overcomes the challenges associated with purifying Skd3 and allows for in depth study of Skd3 activity. Tobacco etch virus (TEV) protease is required in the purification of Skd3. Thus, we also describe how to purify high quality TEV protease for use in the purification of Skd3, other purification protocols, and assays requiring TEV protease.
摘要
Skd3（由人类编码）是一种线粒体AAA+蛋白，由一个N端锚蛋白重复结构域和一个C端HCLR进化枝核苷酸结合结构域组成。由于将该蛋白纯化至高质量和接近均一性存在挑战，Skd3的功能长期以来一直未知。最近，我们将Skd3描述为一种人类线粒体蛋白解聚酶，可溶解线粒体内膜间隙中的蛋白质。本方案克服了与纯化Skd3相关的挑战，并允许对Skd3活性进行深入研究。纯化Skd3需要烟草蚀纹病毒（TEV）蛋白酶。因此，我们还描述了如何纯化高质量的TEV蛋白酶，以用于Skd3的纯化、其他纯化方案以及需要TEV蛋白酶的测定。
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本文引用的文献
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Elife. 2020 Jun 23;9:e55279. doi: 10.7554/eLife.55279.
2
CLPB (caseinolytic peptidase B homolog), the first mitochondrial protein refoldase associated with human disease.CLPB（酪蛋白水解肽酶B同源物），首个与人类疾病相关的线粒体蛋白重折叠酶。
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