期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 53, 期 1, 页码 80-86出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2006.12.004
关键词
MAPK kinase; ERK2; c-Myc
类别
资金
- NIEHS NIH HHS [ES07784] Funding Source: Medline
- NIGMS NIH HHS [R01 GM059802, GM59802] Funding Source: Medline
ERK2 is a mitogen-activated protein kinase (MAPK) that plays pivotal roles in cell signal transduction, where it mediates effects on proliferation and differentiation by growth factors and hormones. An important substrate of ERK2 is the transcription factor c-Myc, which mediates cell cycle progression. The phosphorylation of Ser-62 on c-Myc by ERK2 is thought to contribute to the increased stability of c-Myc during the cell cycle and is thus a critical cellular event. However, the mode of c-Myc recognition by ERK2 is not understood. Early studies by Gupta and Davis concluded that ERK2 specificity determinants are located in residues 1-100 of c-Myc, its activation domain. To pursue both structural and kinetic studies a rapid, but efficient purification method, for the production of the activation domain of c-Myc from an Escherichia coli source, was developed. We chose the minimal number of high-resolution steps to maximize both yield and efficiency without sacrificing purity. Thus, GST-(c-Myc Delta 2-99)-HiS6 was expressed in E coli, and purified using glutathione-agarose affinity chromatography. Cleavage of the GST fusion protein by thrombin and subsequent purification by nickel-agarose affinity chromatography yielded 8 mg of purified (c-MycA2-99)-HiS6 from one liter of LB culture. Rigorous characterization demonstrated that under standard assay conditions (c-Myc Delta 2-99)-HiS6 is phosphorylated by ERK2 with the following Michaelis parameters: k(cat), = 10.4 s(-1), K-M(c-myc) = 57.4 mu M. In summary, a rapid procedure is outlined for the preparation of (c-Myc Delta 2-99)-HiS6 that will be useful for mechanistic and biophysical studies of ERK2. (c) 2006 Elsevier Inc. All rights reserved.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据