Abstract
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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Arabidopsis Proteins / chemistry
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Arabidopsis Proteins / isolation & purification
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Arabidopsis Proteins / pharmacology
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Chromatography, Affinity / methods*
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Escherichia coli / genetics
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Fungal Proteins / chemistry
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Fungal Proteins / isolation & purification
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Fungal Proteins / pharmacology
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Glutathione Transferase / chemistry
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Glutathione Transferase / genetics
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Herpes Simplex Virus Protein Vmw65 / chemistry
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Herpes Simplex Virus Protein Vmw65 / genetics
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Humans
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Protein Structure, Tertiary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / genetics
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification*
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Recombinant Proteins / pharmacology
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TATA-Binding Protein Associated Factors / chemistry
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TATA-Binding Protein Associated Factors / genetics
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TATA-Box Binding Protein / chemistry
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TATA-Box Binding Protein / isolation & purification*
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TATA-Box Binding Protein / pharmacology
Substances
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Arabidopsis Proteins
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Fungal Proteins
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Herpes Simplex Virus Protein Vmw65
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Recombinant Fusion Proteins
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Recombinant Proteins
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TATA-Binding Protein Associated Factors
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TATA-Box Binding Protein
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Glutathione Transferase