小鼠极光激酶A：在大肠杆菌中的表达与纯化

Mouse Aurora A: expression in Escherichia coli and purification.

作者信息

Elling Robert A, Tangonan Bradley T, Penny David M, Smith Jeremy T, Vincent Diana E, Hansen Stig K, O'Brien Tom, Romanowski Michael J

机构信息

Department of Protein Sciences and Structural Biology, Sunesis Pharmaceuticals, Inc., 341 Oyster Point Blvd., South San Francisco, CA 94080, USA.

出版信息

Protein Expr Purif. 2007 Jul;54(1):139-46. doi: 10.1016/j.pep.2007.03.002. Epub 2007 Mar 12.

DOI:10.1016/j.pep.2007.03.002

PMID:17434748

Abstract

Aurora kinases have recently become some of the most intensely pursued oncology targets for the design of small-molecule inhibitors. Most of the active Aurora-A protein variants are currently being expressed from baculoviruses in insect cells, while catalytically impaired proteins can also be generated in and purified from Escherichia coli. In this study we present a method of expressing large quantities of active mouse Aurora-A kinase domain as an N-terminal glutathione-S-transferase fusion protein in bacteria and outline a simple purification method that produces greater than 99% pure protein samples suitable for enzymatic assays and X-ray crystallography. The methods described in this report simplify mouse Aurora-A expression and purification, and may aid in the production of other difficult kinases in prokaryotes.

摘要

极光激酶最近已成为小分子抑制剂设计中最受关注的肿瘤学靶点。目前，大多数活性极光-A蛋白变体是通过杆状病毒在昆虫细胞中表达的，而催化受损的蛋白也可以在大肠杆菌中产生并纯化。在本研究中，我们提出了一种在细菌中表达大量活性小鼠极光-A激酶结构域作为N端谷胱甘肽-S-转移酶融合蛋白的方法，并概述了一种简单的纯化方法，该方法可产生纯度大于99%的适合酶促分析和X射线晶体学的蛋白质样品。本报告中描述的方法简化了小鼠极光-A的表达和纯化，可能有助于在原核生物中生产其他难以表达的激酶。