Purification and Activation of Recombinant p38 Isoforms α, β, γ, and δ

Abstract p38 is a proline-directed serine/threonine kinase that is activated by inflammatory cytokines and cellular stress. At present, four isoforms of p38 have been identified and termed α, β, γ, and δ. We expressed each p38 homolog in Escherichia coli and purified the recombinant isoforms. p38α and C-terminal Flag-tagged p38β were purified by Q-Sepharose fast flow, hydroxyapatite, and Q-Sepharose high-performance chromatography. His-tagged p38γ was purified using Ni 2+ –NTA resin followed by Mono Q chromatography. Glutathione S -transferase–Flag p38δ was purified using M2 affinity agarose and gel-filtration chromatography. Upstream activators of p38, constitutively active (ca) MKK3 and MKK6, were also cloned, purified, and used to activate each p38 isoform. p38 α, γ, and δ were phosphorylated by both MKK6 and caMKK3. p38β was phosphorylated only by MKK6. Mass spectrometry analysis and kinase assays showed that MKK6 was the superior reagent for phosphorylating and activating all p38 isoforms.