Expression of a tyrosine phosphorylated, DNA binding Stat3β dimer in bacteria

FEBS Letters

Becker, Stefan³  Corthals, Garry L.¹  Groner, Bernd²  Müller, Christoph W.³  Aebersold, Ruedi¹ 

[1]Department of Molecular Biotechnology, University of Washington, Box 357730, Seattle, WA 98195-7730, USA

[2]Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg i. Br., Germany

[3]European Molecular Biology Laboratory (EMBL), Grenoble Outstation, c/o ILL, P.O. Box 156, 38042 Grenoble Cedex 9, France

关键词: Stat3β;    Bacterial expression;    In vivo activation;    Tyrosine phosphorylation;    STAT;    signal transducer and activator of transcription;    SH2;    Src homology 2 domain;    PCR;    polymerase chain reaction;    APRE;    acute phase response element;    DTT;    dithiothreitol;    Stat3βtc;    a fragment of Stat3β consisting of amino acid residues 127–722;    HEPES;    N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid);    CAPS;    3-(cyclohexyl-amino)-1-propanesulfonic acid;    PMSF;    phenylmethylsulfonyl fluoride;    MS;    mass spectrometry;    MS/MS;    tandem mass spectrometry;    EGF receptor;    epidermal growth factor receptor;    AspN;    endoproteinase Asp-N;   

DOI  :  10.1016/S0014-5793(98)01543-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The signal transducer and activator of transcription (STAT) proteins deliver signals from the cell membrane to the nucleus. An N-terminally truncated fragment of murine Stat3β, Stat3βtc (127–722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding. Therefore, Stat3βtc was coexpressed with the catalytic domain of the Elk receptor tyrosine kinase. Stat3βtc was quantitatively phosphorylated by this kinase domain. Gel filtration chromatography revealed a Stat3βtc dimer. Y705 was identified as the major phosphorylated residue of Stat3βtc. This corresponds to the tyrosine residue which is phosphorylated by the Janus kinase in vivo. The phosphorylated Stat3βtc specifically bound to DNA binding sites. The described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies.

【 授权许可】

Unknown