Fractionation of RNA polymerase II transcription factors from HeLa cell nuclear extracts by affinity chromatography on "DNA-like" phosphorylated polystyrene.
It was previously shown that phosphorylated cross-linked polystyrene derivatives specifically interacted with anti-DNA antibodies and anti-phospholipid antibodies present in the sera of systemic lupus erythematosus patients. These resins are potential candidates as stationary phases in affinity chromatography. We wondered whether these biospecific resins might allow the fractionation of DNA binding proteins such as RNA polymerase II transcription factors from HeLa cell nuclear extracts. Indeed, these proteins play a major role in gene regulation in mammalian cells and their purification still requires numerous steps. To study the biospecificity of DNA-like phosphorylated polystyrene derivatives, ethanolamine sulfamide crosslinked polystyrene derivatives were phosphorylated at various rates and HeLa cell nuclear extracts were adsorbed on these resins. Adsorbed proteins were eluted with increasing concentrations of aqueous potassium chloride. Collected fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the biological activities of the eluted transcription factors were tested by in vitro transcription assay. Results showed that the elution of transcription factors depended on the substitution rate in phosphoester groups of the resins. It appears that specific interactions were developed between the polymers and the transcription factors. Moreover, the eluted transcription factors kept their biological activity. These results lead us to propose the purification of RNA polymerase II transcription factors using the phosphorylated polystyrene resins as stationary phases.
Imbert
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E
Laboratoire de Recherches sur les Macromolécules, CNRS URA 502, University Paris-Nord, Villetaneuse, France.
Letourneur
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D
Jozefowicz
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M
D001483:Base Sequence; D002467:Cell Nucleus / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D004279:DNA, Viral; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011137:Polystyrenes / Q000737:chemistry*; D012319:RNA Polymerase II / Q000302:isolation & purification* / Q000378:metabolism; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism; D014158:Transcription, Genetic
D004279:DNA, Viral; D011137:Polystyrenes; D014157:Transcription Factors; D012319:RNA Polymerase II