Smith Catherine K, Carr Donna, Mayhood Todd W, Jin Weihong, Gray Kimberly, Windsor William T
Department of Protein Engineering and Biochemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Protein Expr Purif. 2007 Apr;52(2):446-56. doi: 10.1016/j.pep.2006.11.002. Epub 2006 Nov 9.
Kinases exist in either a high or low activity form depending on the phosphorylation state of the activating lip. These two different forms of the same kinase may adopt different conformations that affect not only activity but also inhibitor binding and the ability to crystallize the protein. Therefore, isolation of homogenous preparations of the phosphorylated and non-phosphorylated versions of a kinase is critical for accurate biophysical measurements of activity, stability and ligand binding as well as for protein crystallization. The aim of the present study is the expression, purification and characterization of recombinant human MEK1 protein in both the activated and low-activity states. A baculovirus co-expression system was developed for obtaining high levels of activated, phosphorylated human MEK1 kinase. High-Five cells were co-infected with human MEK1 virus and Raf-BXB, an untagged constitutively active version of Raf which is the activating kinase for MEK1. Unphosphorylated MEK1 was generated by treating MEK1 isolated from High-Five baculovirus expression with lambda-phosphatase. The proteins were characterized by SDS-PAGE, LC-MS, Western blotting, enzymatic activity, and circular dichroism. Previous reports of MEK1 expression and purification yielded lower levels of protein and purity. The yield using High-Five cells was 5mg/L for phosphorylated MEK1 and 10mg/L for unphosphorylated MEK1. For phosphorylated MEK1, the specific activity was 3530U/mg, the IC(50) values for the non-specific kinase inhibitors K252a and K252b were 8 and 47nM, respectively, and the IC(50) for the MEK1 non-ATP competitive inhibitor, PD0325901, was 43nM.
激酶根据激活环的磷酸化状态以高活性或低活性形式存在。同一激酶的这两种不同形式可能具有不同的构象，这不仅会影响活性，还会影响抑制剂结合以及蛋白质结晶的能力。因此，分离激酶的磷酸化和非磷酸化版本的均一制剂对于准确进行活性、稳定性和配体结合的生物物理测量以及蛋白质结晶至关重要。本研究的目的是表达、纯化和表征处于激活状态和低活性状态的重组人MEK1蛋白。开发了一种杆状病毒共表达系统以获得高水平的激活的、磷酸化的人MEK1激酶。将High-Five细胞与人MEK1病毒和Raf-BXB共感染，Raf-BXB是Raf的无标签组成型活性版本，是MEK1的激活激酶。通过用λ-磷酸酶处理从High-Five杆状病毒表达中分离的MEK1来产生未磷酸化的MEK1。通过SDS-PAGE、LC-MS、蛋白质印迹、酶活性和圆二色性对蛋白质进行表征。先前关于MEK1表达和纯化的报道产生的蛋白质水平和纯度较低。使用High-Five细胞时，磷酸化MEK1的产量为5mg/L，未磷酸化MEK1的产量为10mg/L。对于磷酸化的MEK1，比活性为3530U/mg，非特异性激酶抑制剂K252a和K252b的IC(50)值分别为8和47nM，MEK1非ATP竞争性抑制剂PD0325901的IC(50)为43nM。
