One-step affinity purification of recombinant TATA binding proteins utilizing a modular protein interaction partner
- 作者：Dean D. Shooltz ; Glen L. Alberts ; Steven J. Triezenberg
- 关键词：TBP ; Transcription factor ; Promoter ; VP16 ; TAF1 ; TAND2 ; TBP-associated factor ; Transcriptional activation domain
- 刊名：Protein Expression and Purification
- 出版年：2008
- 期刊代码：184_10465928
- 类别：bio
- 出版时间：June 2008
- 卷：59
- 期：2
- 页码：297-301
- 文件大小：325 K
摘要
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.