A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells
| Autor: | Joel P. Mackay, Daniel P. Ryan, Benjamin L. Parker, Hinnerk Saathoff, Jason Low, Mattias Brofelth, Nicholas E. Shepherd, Ana P. G. Silva, Anne Trinh, Sarah R. Webb |
|---|---|
| Rok vydání: | 2015 |
| Předmět: |
Friend of GATA1 (FOG1)
Affinity label Molecular Sequence Data Clinical Biochemistry Pharmaceutical Science Peptide Biochemistry Catalysis Chromatin remodeling Mice 03 medical and health sciences Affinity Reagent Drug Discovery Tumor Cells Cultured Animals Nucleosome Amino Acid Sequence Molecular Biology Peptide sequence Chromatography High Pressure Liquid 030304 developmental biology Medicine(all) chemistry.chemical_classification 0303 health sciences biology 030302 biochemistry & molecular biology Organic Chemistry Affinity Labels Affinity purification Nucleosome remodeling and deacetylase complex peptides Mi-2/NuRD complex chemistry Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Molecular Medicine Electrophoresis Polyacrylamide Gel Multi-protein complex Peptides Linker Mi-2 Nucleosome Remodeling and Deacetylase Complex |
| Zdroj: | Bioorganic & Medicinal Chemistry. 23:960-965 |
| ISSN: | 0968-0896 |
| DOI: | 10.1016/j.bmc.2015.01.023 |
| Popis: | We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1–15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST–FOG1(1–45). |
| Databáze: | OpenAIRE |
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