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Oak-Ridge centrifugation tubes, 50 ml

Oak-Ridge离心管，50 ml
Company: Carl Roth
Catalog#: PK03.1
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In vitro Deneddylation Assay
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Date:
2016-03-20
[Abstract]  Nedd8 is a small ubiquitin-like protein (9 kDa) covalently attached to a conserved lysine residue of a cullin protein which is part of cullin-RING ligases (CRLs). CRLs are major E3 ligases important for protein ubiquitination in the ubiquitin-proteasome pathway (UPP). The activity of CRLs is regulated by cycles of neddylation (CulA-N8, ~98 kDa) and deneddylation (CulA ~89 kDa). The COP9 signalosome (CSN) and Deneddylase A (DenA) are capable of cleaving the isopeptide bond between Nedd8 and CullinA. In contrast to the single protein DenA, CSN is an eight subunit multiprotein complex. Protein crude extracts of different Aspergillus nidulans csn deletion strains were mixed with recombinant CSN subunits expressed and purified from Escherichia coli (E. coli). Western ... [摘要]  Nedd8是共价连接到作为cullin-RING连接酶（CRL）的一部分的cullin蛋白的保守赖氨酸残基的小的遍在蛋白样蛋白（9kDa）。 CRL是对泛素 - 蛋白酶体途径（UPP）中的蛋白质泛素化重要的主要E3连接酶。 CRL的活性通过脱甲基化（CulA-N8，〜98kDa）和脱甲基化（CulA〜89kDa）的循环调节。 COP9信号体（CSN）和丁二酸酶A（DenA）能够切割Nedd8和CullinA之间的异肽键。与单一蛋白DenA相反，CSN是八亚基多蛋白复合物。将不同的构巢曲霉csn 缺失菌株的蛋白质粗提物与从大肠杆菌（大肠杆菌）表达和纯化的重组CSN亚基混合。使用抗CulA或抗Nedd8抗体的Western杂交实验可以显示Nedd化化合物与Denedd化CulA的比率。使用deneddylation测定，我们可以显示CsnE是在体外连接7-亚基预组装的CSN的最后一个亚基，然后CSN可以通过金属蛋白酶亚基CsnE执行cullin deneddylation。该测定法是一种快速且非昂贵的方法，其显现了用于脱蛋白的蛋白质的酶活性。它还可用于测试除去来自构巢曲霉（构巢曲霉）或其他生物体中的底物的翻译后修饰的其它藻肽的活性。

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