Overexpression and Purification of Eukaryotic Transcription Factors as Glutathione-S-Transferase Fusions in E. coli

The ability to express recombinant proteins at high level in bacteria has led to dramatic increases in our understanding of protein structure and function in recent years. These techniques have provided the means to isolate the substantial quantities of recombinant protein required for both X-ray crystallographic and NMR-based structure determinations on what is becoming a nearly routine basis. The opportunity now also exists for the construction of recombinant mutants that are providing much greater insight into protein structure/function relationships. Moreover, comparisons between the activities of purified recombinant proteins and proteins purified from natural sources have often led to the discovery of previously unidentified molecules that are required for full activity of the protein under investigation. A recent example of this phenomenon has been the discovery of coactivator proteins that are internal components of the eukaryotic RNA polymerase II transcription factor TFIID (1 ).