通过固定化轴蛋白亲和层析法纯化细胞周期蛋白依赖性激酶1（CK1）

Purification of CK1 by affinity chromatography on immobilised axin.

作者信息

Reinhardt Jens, Ferandin Yoan, Meijer Laurent

机构信息

Amyloids and Cell Division Cycle, Station Biologique de Roscoff, CNRS, Place Georges Teissier, 29680 Roscoff, France.

出版信息

Protein Expr Purif. 2007 Jul;54(1):101-9. doi: 10.1016/j.pep.2007.02.020. Epub 2007 Mar 12.

DOI:10.1016/j.pep.2007.02.020

PMID:17434749

Abstract

Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.

摘要

酪蛋白激酶1（CK1）家族成员参与多种生理过程的调节，如发育和昼夜节律，以及癌症和阿尔茨海默病等疾病。从这个角度来看，CK1家族成员是潜在化疗的有趣靶点。我们在此描述一种通过固定在轴蛋白片段上的亲和色谱快速高效纯化CK1的方法。轴蛋白是一种支架蛋白，它与多种蛋白质相互作用，其中包括APC、GSK-3、β-连环蛋白、CK1α、δ和ε以及PP2A。在大肠杆菌中产生了一种带有GST标签的轴蛋白肽（残基495 - 684），它要么固定在谷胱甘肽琼脂糖珠上，要么纯化后固定在CNBr活化的琼脂糖4B上。发现这些“GST - 轴蛋白”基质能选择性地结合来自猪脑的天然CK1α和CK1ε。亲和纯化的酶显示出高激酶活性。这种单步纯化方法为高效纯化大量活性天然CK1用于筛选目的提供了一种便捷工具。这种单步纯化方法还为跟踪不同生理环境下轴蛋白结合的CK1同工型α、δ和ε的状态（蛋白质水平、同工型组成、激酶活性）提供了一种便捷工具。