Phosphorylation of two eukaryotic transcription factors, Jun dimerization protein 2 and activation transcription factor 2, in Escherichia coli by Jun N-terminal kinase 1
Phosphorylation of two eukaryotic transcription factors, Jun dimerization protein 2 and activation transcription factor 2, in Escherichia coli by Jun N-terminal kinase 1
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DOI：
10.1016/j.ab.2008.01.038
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发表时间：
2008-05-01
影响因子：
2.9
通讯作者：
Yokoyama, Kazunarl K.
中科院分区：
文献类型：
--
作者：
Murata, Takehide;Shinozuka, Yoriko;Yokoyama, Kazunarl K.
重组真核蛋白经常在大肠杆菌中产生，并且此类蛋白经常用于体外生物化学研究。然而，以这种方式产生的蛋白质在细菌细胞中合成期间不会被化学修饰，例如通过磷酸化、乙酰化、甲基化、糖基化或遍在蛋白化。我们构建了用于在大肠杆菌中表达人Jun N-末端激酶1（JNK 1）、小鼠Aurora激酶B（Aurkb）和P/CAF的历史乙酰转移酶（HAT）结构域的载体。这些表达载体包括p15 A的复制起点和pBR 322或Co 1 E1的复制起点。将这些表达载体在E.在大肠杆菌中，我们能够通过同时表达的人JNK 1的作用磷酸化小鼠和人Jun二聚化蛋白2（JDP 2）和人激活转录因子2（ATF-2）。此外，小鼠组蛋白H3的尾部区域分别被Aurkb和P/CAF的HAT结构域磷酸化和乙酰化。我们还观察到当ATF-2被磷酸化时，ATF-2与JDP 2的相互作用被阻止。我们在大肠杆菌中生产酶修饰蛋白的表达系统。大肠杆菌中的化学修饰的真核生物蛋白质的体外生物化学研究具有广泛的应用前景。(c)2008年爱思唯尔公司All rights reserved.
Recombinant eukaryotic proteins are frequently produced in Escherichia coli and such proteins are often used for biochemical studies in vitro. However, proteins produced in this way are not modified chemically, for example, by phosphorylation, acetylation, methylation, surnoylation, or ubiquitination, during their synthesis in bacterial cells. We constructed vectors for expression in E coli of human Jun N-terminal kinase 1 (JNK1), mouse Aurora kinase B (Aurkb), and the historic acetyltransferase (HAT) domain of P/CAF. These expression vectors included the origin of replication of p15A and the origin of replication of pBR322 or Co1E1. Using these expression vectors in E. coli, we were able to phosphorylate mouse and human Jun dimerization protein 2 (JDP2) and human activation transcription factor 2 (ATF-2) by the action of human JNK1 that was expressed simultaneously. Moreover, the tail region of mouse histone H3 was phosphorylated and acetylated, respectively, by Aurkb and by the HAT domain of P/CAF. We also observed that the interaction of ATF-2 with JDP2 was prevented when ATF-2 was phosphorylated. Our expression systems for production of enzyme-modified proteins in E. coli should be widely applicable and useful for biochemical studies of chemically modified eukaryotic proteins in vitro. (c) 2008 Elsevier Inc. All rights reserved.