title: Efficient overexpression and purification of active full-length human transcription factor Yin Yang 1 in Escherichia coli.
abstract: 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.
authors.affiliation: Department of Physical Biochemistry, Jagiellonian University, Gronostajowa 7, Kraków, Poland.
authors.forename: Filip M
authors.initials: FM
authors.lastname: Golebiowski
authors.forename: Andrzej
authors.initials: A
authors.lastname: Górecki
authors.forename: Piotr
authors.initials: P
authors.lastname: Bonarek
authors.forename: Marta
authors.initials: M
authors.lastname: Dziedzicka-Wasylewska
mesh_terms: D001665:Binding Sites; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli; D005454:Fluorescence Polarization; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D058849:Protein Refolding; D055550:Protein Stability; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D050986:YY1 Transcription Factor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011994:Recombinant Proteins; D050986:YY1 Transcription Factor; C494698:YY1 protein, human; D006639:Histidine; D004247:DNA