Efficient overexpression and purification of active full-length human transcription factor Yin Yang 1 in Escherichia coli

The Yin Yang 1 protein is a zinc finger transcription factor involved in the regulation of diverse cellular processes through DNA and protein–protein interactions. Here we present an improved method for the expression and purification of the human full-length YY1 protein from Escherichia coli. The p...

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Bibliographic Details
Published in:Protein expression and purification 2011-06, Vol.77 (2), p.198-206
Main Authors: Golebiowski, Filip M., Górecki, Andrzej, Bonarek, Piotr, Dziedzicka-Wasylewska, Marta
Format: Article
Language:English
Subjects:
Binding Sites
Chromatography, Affinity
Cloning, Molecular
Disulfide reduction
DNA - metabolism
DNA-affinity purification
Electrophoretic Mobility Shift Assay
Escherichia coli
Fluorescence Polarization
Gene Expression
Histidine - metabolism
Humans
Inclusion Bodies - genetics
Inclusion Bodies - metabolism
mRNA secondary structure
Oligopeptides - metabolism
Plasmids - genetics
Plasmids - metabolism
Protein Binding
Protein Denaturation
Protein Engineering - methods
Protein Refolding
Protein Stability
Recombinant Proteins - genetics
Recombinant Proteins - isolation & purification
Recombinant Proteins - metabolism
YY1 Transcription Factor - genetics
YY1 Transcription Factor - isolation & purification
YY1 Transcription Factor - metabolism
Zinc Fingers
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Summary:The Yin Yang 1 protein is a zinc finger transcription factor involved in the regulation of diverse cellular processes through DNA and protein–protein interactions. Here we present an improved method for the expression and purification of the human full-length YY1 protein from Escherichia coli. The protein was first purified using denaturing conditions, refolded using optimized conditions and then purified using a DNA-affinity column to ⩾95% purity; this process provided a high final yield and highly active protein. The protein was active in EMSA and the fluorescence anisotropy assays. The protein retained its full activity and its initial concentration for several months when stored at −80 °C. Thus, we have obtained YY1 protein with levels of activity and concentration that are suitable for spectroscopic and other biochemical studies.
ISSN:1046-5928
1096-0279
DOI:10.1016/j.pep.2011.01.016