The c-Jun N-terminal kinase (INK) 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 JNK1 beta 1, which is most suitable for biochemical and biophysical characterization. Codon harmonization of the JNK1 beta 1 gene was used as a precautionary measure toward increasing the soluble overexpression of the kinase. While JNK1 beta 1 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. coil as thioredoxin-His(6)-tagged proteins and purified using urea refolding and Ni2+-IMAC, respectively. Activation of JNK1 beta 1 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 JNK1 beta 1. Activated JNK1 beta 1 was thereafter able to phosphorylate ATF2 with high catalytic efficiency. (C) 2012 Elsevier Inc. All rights reserved.