Expression of a tyrosine phosphorylated, DNA binding Stat3beta dimer in bacteria.
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 Stat3beta, Stat3betatc (127-722), was produced in bacteria. STAT proteins must be specifically phosphorylated at a single tyrosine residue for dimerization and DNA binding. Therefore, Stat3betatc was coexpressed with the catalytic domain of the Elk receptor tyrosine kinase. Stat3betatc was quantitatively phosphorylated by this kinase domain. Gel filtration chromatography revealed a Stat3betatc dimer. Y705 was identified as the major phosphorylated residue of Stat3betatc. This corresponds to the tyrosine residue which is phosphorylated by the Janus kinase in vivo. The phosphorylated Stat3betatc specifically bound to DNA binding sites. The described protocol allows the production of large amounts of activated protein for biochemical and pharmaceutical studies.
Becker
S
S
European Molecular Biology Laboratory, Grenoble, France.
Corthals
G L
GL
Aebersold
R
R
Groner
B
B
Müller
C W
CW
D000209:Acute-Phase Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019281:Dimerization; D004926:Escherichia coli; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D050796:STAT3 Transcription Factor; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000209:Acute-Phase Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D050796:STAT3 Transcription Factor; C494087:Stat3 protein, mouse; D015534:Trans-Activators; D019000:Phosphotyrosine