Protein Expression and Purification, Vol.59, No.2, 297-301, 2008
One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner
We describe a rapid and effective procedure for purifying recombinant eukaryotic TATA binding protein (TBP) from Escherichia coli. The method employs an affinity ligand comprising glutathione-S-transferase fused to the carboxyl-terminal activation domain of the transcriptional activator VP16 and an amino-terminal domain (TAND2) of the yeast TBP-associated factor TAF1. TBP can be purified without the need for extrinsic affinity tags, subsequent proteolysis, or downstream clean-up steps. This TBP purification process is rapid (requiring about 4 h after bacterial harvest) and does not require sophisticated chromatographic equipment. The resulting material is monodisperse, structured, and functionally active. We demonstrate the efficacy of this method for purifying recombinant full-length or TBP core fragments encoded by yeast, humans and Arabidopsis. (C) 2008 Elsevier Inc. All rights reserved.
Keywords:TBP;transcription factor;promoter;VP16;TAF1;TAND2;TBP-associated factor;transcriptional activation domain