B. Chatton et al., EUKARYOTIC GST FUSION VECTOR FOR THE STUDY OF PROTEIN-PROTEIN ASSOCIATIONS IN-VIVO - APPLICATION TO INTERACTION OF ATFA WITH JUN AND FOS, BioTechniques, 18(1), 1995, pp. 142-145
We describe a multipurpose eukaryotic expression vector that incorpora
tes the following features: restriction sites for in-frame insertion o
f cDNAs of interest between sequences encoding the glutathione-S-trans
ferase (GST) and an oligohistidine elements, allowing expression of th
e corresponding fusion proteins; a phosphorylation site for protein ki
nase A for in vitro labeling of the fusion protein; a T7 promoter for
in vitro transcription and subsequent translation; and signals for sin
gle-stranded DNA production in bacteria. We have used this vector to d
emonstrate the formation in vivo of complexes between the transcriptio
n factor ATFa, a member of the family of ATF/CRE binding proteins, and
the c-Jun or c-Fos proteins. Such interactions could be detected in c
rude extracts from cells transfected with vectors expressing the GST-A
TFa fusion protein, as well as the c-Jun or c-Fos proteins. Complexes
containing both ATFa and either c-Jun or c-Fos were specifically retai
ned on glutathione (GSH)-agarose bends as revealed by immunoblot analy
ses. We also show that the leucine zipper do,nain of ATFa is essential
for this interaction.