The c-Jun N-terminal kinase (JNK) pathway forms part of the mitogen-activated 
protein kinase (MAPK) signaling pathways comprising a sequential three-tiered 
kinase cascade. Here, an upstream MAP3K (MEKK1) phosphorylates and activates a 
MAP2K (MKK4 and MKK7), which in turn phosphorylates and activates the MAPK, JNK. 
The C-terminal kinase domain of MEKK1 (MEKK-C) is constitutively active, while 
MKK4/7 and JNK are both activated by dual phosphorylation of S/Y, and T/Y 
residues within their activation loops, respectively. While improvements in the 
purification of large quantities of active JNKs have recently been made, 
inadequacies in their yield, purity, and the efficiency of their phosphorylation 
still exist. We describe a novel and robust method that further improves upon 
the purification of large yields of highly pure, phosphorylated JNK1β1, which is 
most suitable for biochemical and biophysical characterization. Codon 
harmonization of the JNK1β1 gene was used as a precautionary measure toward 
increasing the soluble overexpression of the kinase. While JNK1β1 and its 
substrate ATF2 were both purified to >99% purity as GST fusion proteins using 
GSH-agarose affinity chromatography and each cleaved from GST using thrombin, 
constitutively-active MEKK-C and inactive MKK4 were separately expressed in E. 
coli as thioredoxin-His(6)-tagged proteins and purified using urea refolding and 
Ni(2+)-IMAC, respectively. Activation of JNK1β1 was then achieved by 
successfully reconstituting the JNK MAPK activation cascade in vitro; MEKK-C was 
used to activate MKK4, which in turn was used to efficiently phosphorylate and 
activate large quantities of JNK1β1. Activated JNK1β1 was thereafter able to 
phosphorylate ATF2 with high catalytic efficiency.