1 Prevention of MKK6-Dependent Activation by Binding to p38α MAP Kinase: Supporting Information Jane E. Sullivan, Geoffrey A. Holdgate, Douglas Campbell, David Timms, Stefan Gerhardt, Jason Breed, Alexander L. Breeze, Alun Bermingham, Richard A. Pauptit, Richard A. Norman, Kevin J. Embrey, Jon Read, Wendy S. VanScyoc and Walter H. J. Ward * AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK * Author for correspondence. E-mail: walter.ward@astrazeneca.com. Tel: ++ 44 (0)1625 515998 Expression, labelling and purification of human recombinant p38 α. DNA constructs encoding p38α (residue 2 to C-terminus), with either an N- terminal-6His-16j tag (MGSSHHHHHHLVPRGSH), or an N-terminal-cmyc- 6His tag (MGSEQKLISEEDLNAHHHHHH), were cloned into a pT73.3 vector (1), using the Xho1 and Ase1 restriction enzyme sites. The cmyc-6His construct was used for NMR and in assays for enzyme activity and inhibitor binding. The 6His-16j construct was used for crystallography. Proteins were expressed in Escherichia coli BL21(DE3) strain MSD3716 in L-broth plus 10 μg/ml tetracyclin at 30 °C until an A 550 of 0.5 was reached. Expression was induced by 0.4 mM isopropyl β-D-thiogalactopyranoside and the bacteria harvested 5 h later. Cell pastes were frozen and stored at -80 °C. Cells were lysed by sonication (cmyc-6His-p38α) or emulsiflex (6His- 16j-p38α) in 20 mM Tris pH7.5, 0.5 M NaCl, 5 mM imidazole, 0.1 mM