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Published on: October 25, 2014

Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes

David M Rees¹, Oliver Willhoft¹, Chia-Liang Lin¹

¹Section of Structural Biology, Imperial College London, London, United Kingdom.

Methods in Enzymology

|July 3, 2017

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

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