Reference - Detail
|Author||Murata T, Shinozuka Y, Obata Y, Yokoyama KK.|
|Title||Phosphorylation of two eukaryotic transcription factors, Jun dimerization protein 2 and activation transcription factor 2, in Escherichia coli by Jun N-terminal kinase 1.|
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, sumoylation, 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 histone acetyltransferase (HAT) domain of P/CAF. These expression vectors included the origin of replication of p15A and the origin of replication of pBR322 or ColE1. 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.
|MeSH||Activating Transcription Factor 2 / genetics Activating Transcription Factor 2 / metabolism* Animals Escherichia coli / genetics Humans Mice Mitogen-Activated Protein Kinase 8 / genetics Mitogen-Activated Protein Kinase 8 / metabolism* Phosphorylation Protein Binding Recombinant Proteins / metabolism* Repressor Proteins / genetics Repressor Proteins / metabolism*|
|WOS Category||CHEMISTRY, ANALYTICAL BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY|
|DNA material||pACYC184 pGST-mmJDP2_full (RDB04757) pGST-mmJDP2_T148A (RDB04778) pET_His-hsJDP2 (RDB06216) pET_HisFLAG-hsATF-2 (RDB06321) pT7Tet1 (RDB06211) NIA 15K Mouse cDNA Clone (RDB05723) pEThsPCAF-HAT (RDB06085) pEThsJNK1 (RDB06088) pT7Cam_hsJNK1 (RDB06089) pGex_mmhist1h3f (RDB06124) pT7Cam_hsPCAF HAT (RDB06125) pmmAurkb (RDB06213) pT7Tet_mmAurkb (RDB06214) pET_HisFLAG-hsJDP2 (RDB06215) pET_His-hsJDP2 (RDB06216) pET_HisFLAG-hsATF-2 (RDB06321) pGEX-mmH3_tail (RDB06322)|
|Prokaryotes E. coli|