白细胞介素-2酪氨酸激酶的细菌表达与纯化：伴侣蛋白杂质的一步分离
Bacterial expression and purification of interleukin-2 tyrosine kinase: single step separation of the chaperonin impurity.
作者信息
Joseph Raji E, Andreotti Amy H
机构信息
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 4208 MBB, Ames, IA 50011, USA.
出版信息
Protein Expr Purif. 2008 Aug;60(2):194-7. doi: 10.1016/j.pep.2008.04.001. Epub 2008 Apr 11.
Abstract
Biochemical and biophysical characterization of kinases requires large quantities of purified protein. Here, we report the bacterial expression and purification of active Itk kinase domain (a Tec family kinase) using ArcticExpress cells that co-express the chaperonin system Cpn60/10 from Oleispira antarctica. We describe a simple one step MgCl2/ATP/KCl incubation procedure to remove the co-purifying chaperonin impurity. Chaperonin co-purification is a common problem encountered during protein purification and the simple incubation step described here completely overcomes this problem. The approach targets the chaperonin system rather than the protein of interest and is therefore widely applicable to other protein targets.
摘要
激酶的生化和生物物理特性表征需要大量纯化的蛋白质。在此，我们报告了使用共表达来自南极油螺旋菌伴侣蛋白系统Cpn60/10的北极快车细胞，对活性Itk激酶结构域（一种Tec家族激酶）进行细菌表达和纯化。我们描述了一种简单的一步MgCl2/ATP/KCl孵育程序，以去除共纯化的伴侣蛋白杂质。伴侣蛋白共纯化是蛋白质纯化过程中常见的问题，此处描述的简单孵育步骤完全克服了这个问题。该方法针对的是伴侣蛋白系统而非目标蛋白，因此广泛适用于其他蛋白质靶点。
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