Expression and purification of phosphorylated and non-phosphorylated human MEK1.
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.
Smith
Catherine K
CK
Department of Protein Engineering and Biochemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA. catherine.smith15@spcorp.com
Carr
Donna
D
Mayhood
Todd W
TW
Jin
Weihong
W
Gray
Kimberly
K
Windsor
William T
WT
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D015870:Gene Expression / Q000502:physiology*; D006801:Humans; D007313:Insecta / Q000166:cytology; D048369:MAP Kinase Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism
D012694:Serine; D048369:MAP Kinase Kinase 1; C482125:MAP2K1 protein, human