Eukaryotic GST fusion vector for the study of protein-protein associations in vivo: application to interaction of ATFa with Jun and Fos

Biotechniques. 1995 Jan;18(1):142-5.

Abstract

We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine element, allowing expression of the corresponding fusion proteins; a phosphorylation site for protein kinase A for in vitro labeling of the fusion protein; a T7 promoter for in vitro transcription and subsequent translation; and signals for single-stranded DNA production in bacteria. We have used this vector to demonstrate the formation in vivo of complexes between the transcription 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 crude extracts from cells transfected with vectors expressing the GST-ATFa 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 retained on glutathione (GSH)-agarose beads as revealed by immunoblot analyses. We also show that the leucine zipper domain of ATFa is essential for this interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factors
  • Amino Acid Sequence
  • Base Sequence
  • Blood Proteins / metabolism*
  • Genetic Vectors*
  • Glutathione Transferase / genetics*
  • Molecular Sequence Data
  • Proto-Oncogene Proteins c-fos / metabolism*
  • Proto-Oncogene Proteins c-jun / metabolism*
  • Recombinant Fusion Proteins / metabolism*
  • Transcription Factors / metabolism*
  • Transfection

Substances

  • Activating Transcription Factors
  • Blood Proteins
  • Proto-Oncogene Proteins c-fos
  • Proto-Oncogene Proteins c-jun
  • Recombinant Fusion Proteins
  • Transcription Factors
  • Glutathione Transferase

Associated data

  • GENBANK/X78316