Purification and Activation of Recombinant p38 Isoforms , &bgr;, &ggr;, and &dgr;

Author： Keesler G.A. Bray J. Hunt J. Johnson D.A. Gleason T. Yao Z. Wang S-W. Parker C. Yamane H. Cole C. Lichenstein H.S.

Publisher： Academic Press

ISSN： 1046-5928

Source： Protein Expression and Purification, Vol.14, Iss.2, 1998-11, pp. : 221-228

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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 , &bgr;, &ggr;, and &dgr;. We expressed each p38 homolog in Escherichia coli and purified the recombinant isoforms. p38 and C-terminal Flag-tagged p38&bgr; were purified by Q-Sepharose fast flow, hydroxyapatite, and Q-Sepharose high-performance chromatography. His-tagged p38&ggr; was purified using Ni²⁺–NTA resin followed by Mono Q chromatography. Glutathione S-transferase–Flag p38&dgr; 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 &agr;, &ggr;, and &dgr; were phosphorylated by both MKK6 and caMKK3. p38&bgr; 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.