Purification and activation of recombinant p38 isoforms α, β, γ, and δ
Purification and activation of recombinant p38 isoforms α, β, γ, and δ
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DOI：
10.1006/prep.1998.0947
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发表时间：
1998-11-01
影响因子：
1.6
通讯作者：
Lichenstein, HS
中科院分区：
文献类型：
--
作者：
Keesler, GA;Bray, J;Lichenstein, HS
p38 是一种脯氨酸导向的丝氨酸/苏氨酸激酶，可被炎症细胞因子和细胞应激激活。目前，p38 的四种亚型已被鉴定并命名为 α、β、γ 和 δ。我们在大肠杆菌中表达了每个 p38 同源物并纯化了重组异构体。 p38 α 和 C 末端 Flag 标记的 p38 β 通过 Q-Sepharose fast how、羟基磷灰石和 Q-Sepharose 高效色谱纯化。使用 Ni2+-NTA 树脂和 Mono Q 色谱法纯化带有 His 标签的 p38 γ。使用M2亲和琼脂糖和凝胶过滤层析纯化谷胱甘肽S-转移酶-Flag p38 delta。 p38 的上游激活剂，即组成型活性 (ca) MKK3 和 MKK6，也被克隆、纯化，并用于激活每种 p38 同工型。 p38 α、γ 和 δ 被 MKK6 和 caMKK3 磷酸化。 p38 beta 仅被 MKK6 磷酸化。质谱分析和激酶测定表明 MKK6 是磷酸化和激活所有 p38 同工型的优质试剂。 (C) 1998 年学术出版社。
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 alpha, beta, gamma, and delta. We expressed each p38 homolog in Escherichia coli and purified the recombinant isoforms. p38 alpha and C-terminal Flag-tagged p38 beta were purified by Q-Sepharose fast how, hydroxyapatite, and Q-Sepharose high-performance chromatography. His-tagged p38 gamma was purified using Ni2+-NTA resin followed by Mono Q chromatography. Glutathione S-transferase-Flag p38 delta 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 alpha, gamma, and delta were phosphorylated by both MKK6 and caMKK3. p38 beta 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. (C) 1998 Academic Press.