Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes

Methods Enzymol. 2017:592:27-47. doi: 10.1016/bs.mie.2017.03.015. Epub 2017 Apr 17.

Abstract

We have developed a novel system to facilitate the rapid and easy cloning of multiple genes (>10) in under a week. Using this system we have been able to successfully clone, overexpress, and purify a number of large multimeric proteins from insect cells, including the chromatin remodeling complexes SWR1 and INO80. Using Förster resonance energy transfer (FRET)-based assays we have demonstrated that our overexpressed enzymes have activities comparable to those purified from sources where the proteins are expressed under their endogenous promoters.

Keywords: Chromatin remodeling; DNA repair; Histone exchange; Insect cell expression; Multisubunit complexes; Nuclear actin.

MeSH terms

  • ATPases Associated with Diverse Cellular Activities
  • Adenosine Triphosphatases / genetics*
  • Adenosine Triphosphatases / metabolism
  • Animals
  • Chromatin Assembly and Disassembly*
  • Cloning, Molecular / methods*
  • DNA Helicases / genetics*
  • DNA Helicases / metabolism
  • DNA-Binding Proteins
  • Fluorescence Resonance Energy Transfer / methods
  • Humans
  • Insecta / cytology
  • Insecta / genetics
  • Multigene Family
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism

Substances

  • DNA-Binding Proteins
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • Swr1 protein, S cerevisiae
  • ATPases Associated with Diverse Cellular Activities
  • DNA Helicases
  • INO80 protein, human