Waas W F, Dalby K N
Division of Medicinal Chemistry, University of Texas at Austin 78712, USA.
Protein Expr Purif. 2001 Oct;23(1):191-7. doi: 10.1006/prep.2001.1491.
ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects on proliferation and differentiation by growth factors and hormones. While its cellular function has been under intense scrutiny since its initial discovery nearly 15 years ago, little progress has been made in understanding its kinetic mechanism. Such an understanding is important for the development of potent and specific inhibitors. A contributory factor has been the lack of a protein substrate suitable for rigorous mechanistic studies. Here we report the expression, purification, and characterization of the N-terminus (residues 1 through 138) of the transcription factor Ets-1, an excellent model substrate for ERK2 mechanistic studies. (His(6)-tagged)Ets-1(1-138) was expressed in Escherichia coli and rapidly purified in two steps by nickel-agarose-affinity chromatography, followed by high-resolution Mono-Q anion-exchange chromatography. A yield of 60 mg of the purified protein per liter of culture was obtained and could be stored conveniently at -80 degrees C in water. Rigorous characterization demonstrated that under the assay conditions, (His(6)-tagged)Ets-1(1-138) is exclusively phosphorylated on residue Thr-38 by ERK2 with the following Michaelis parameters: k(cat) = 17 s(-1), K(ATP)(m) = 140 microM, K(ATP)(i) = 68 microM, K(Ets-1)(m) = 19 microM, and K(Ets-1)(i) = 9.3 microM.
ERK2属于丝裂原活化蛋白激酶亚家族，在细胞信号转导中起关键作用，它通过生长因子和激素介导对细胞增殖和分化的影响。自近15年前首次发现以来，其细胞功能一直受到密切关注，但在理解其动力学机制方面进展甚微。这种理解对于开发强效和特异性抑制剂很重要。一个促成因素是缺乏适合进行严格机制研究的蛋白质底物。在此，我们报告了转录因子Ets-1的N端（第1至138位氨基酸残基）的表达、纯化和特性鉴定，它是用于ERK2机制研究的优秀模型底物。（His(6)标签化的）Ets-1(1-138)在大肠杆菌中表达，并通过镍琼脂糖亲和色谱两步快速纯化，随后进行高分辨率Mono-Q阴离子交换色谱。每升培养物可获得60毫克纯化蛋白，并且可以方便地在-80℃水中储存。严格的特性鉴定表明，在测定条件下，（His(6)标签化的）Ets-1(1-138)仅在第38位苏氨酸残基上被ERK2磷酸化，其米氏参数如下：k(cat) = 17 s(-1)，K(ATP)(m) = 140 microM，K(ATP)(i) = 68 microM，K(Ets-1)(m) = 19 microM，以及K(Ets-1)(i) = 9.3 microM。
