A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

Hinnerk Saathoff; Mattias Brofelth; Anne Trinh; Benjamin L Parker; Daniel P Ryan; Jason K K Low; Sarah R Webb; Ana P G Silva; Joel P Mackay; Nicholas E Shepherd

doi:10.1016/j.bmc.2015.01.023

A peptide affinity reagent for isolating an intact and catalytically active multi-protein complex from mammalian cells

Bioorg Med Chem. 2015 Mar 1;23(5):960-5. doi: 10.1016/j.bmc.2015.01.023. Epub 2015 Jan 22.

Affiliations

¹ School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia.
² John Curtin School of Medical Research, The Australian National University, Canberra, ACT 0200, Australia.
³ School of Molecular Bioscience, The University of Sydney, Sydney, NSW 2006, Australia. Electronic address: nicholas.shepherd@sydney.edu.au.

PMID: 25678017
DOI: 10.1016/j.bmc.2015.01.023

Abstract

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).

Keywords: Affinity purification; Friend of GATA1 (FOG1); Multi-protein complex; Nucleosome remodeling and deacetylase complex peptides.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Affinity Labels*
Amino Acid Sequence
Animals
Catalysis
Chromatography, High Pressure Liquid
Electrophoresis, Polyacrylamide Gel
Mi-2 Nucleosome Remodeling and Deacetylase Complex / isolation & purification*
Mi-2 Nucleosome Remodeling and Deacetylase Complex / metabolism*
Mice
Molecular Sequence Data
Peptides / chemistry*
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Tumor Cells, Cultured

Substances

Affinity Labels
Peptides
Mi-2 Nucleosome Remodeling and Deacetylase Complex