Improved expression, purification, and crystallization of p38α MAP kinase

p38alpha mitogen-activated protein (MAP) kinase is widely expressed in many mammalian tissues and is activated as a part of signal transduction cascades that respond to inflammatory stimuli. The activation of p38 is known to trigger various biological effects, including cell death, differentiation, and proliferation. The central role played by p38alpha in cellular signaling events, including those that control a wide range of inflammatory and autoimmune diseases, makes it an attractive drug target. To develop optimized small molecule therapeutics targeting p38alpha different techniques must be employed for the detailed biochemical, biophysical, and structural characterization of the interactions of p38alpha with lead compounds. These methods typically require large quantities of highly purified p38alpha protein. We describe here an improved expression and purification method for recombinant p38alpha production that reproducibly yields over 70 mg of highly purified protein per liter of shake flask bacterial culture. This yield is significantly higher than that previously reported for p38alpha production in Escherichia coli. We achieved a significant increase in soluble p38alpha protein expression by using the genetically modified E eoli strain BL21 DE3 Rosetta, which is optimized for expression of eukaryotic proteins with codons rarely used in E. coli. The p38alpha protein was purified to near homogeneity using a simple two-step procedure including nickel-chelating Sepharose chromatography followed by anion-exchange chromatography using MonoQ resin. Purified p38alpha was characterized using the standard commercially available small molecule inhibitor SB-203580. The binding association and dissociation rate constants determined by Biacore are in excellent agreement with previously reported values. The purified p38ot protein was efficiently activated by MKK6 kinase to yield phosphorylated p38alpha. Purified p38alpha protein was also successfully crystallized, producing crystals diffracting to 1.9 Angstrom, exceeding the highest resolution for p38alpha reported in the Protein DataBank. The simplicity and efficiency of this approach should prove useful for many laboratories that are interested in production of p38alpha for biochemical and biophysical studies and structure-based drug design. (C) 2004 Elsevier Inc. All rights reserved.