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 4h 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.
Shooltz
Dean D
DD
Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA.
Alberts
Glen L
GL
Triezenberg
Steven J
SJ
D029681:Arabidopsis Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000737:chemistry / Q000235:genetics; D006801:Humans; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D035381:TATA-Binding Protein Associated Factors / Q000737:chemistry / Q000235:genetics; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology
D029681:Arabidopsis Proteins; D005656:Fungal Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D005982:Glutathione Transferase