1. Production and Assay of Recombinant Multisubunit Chromatin Remodeling Complexes.
- Author
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Rees DM, Willhoft O, Lin CL, Bythell-Douglas R, and Wigley DB
- Subjects
- ATPases Associated with Diverse Cellular Activities, Adenosine Triphosphatases metabolism, Animals, 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 metabolism, Saccharomyces cerevisiae Proteins metabolism, Adenosine Triphosphatases genetics, Chromatin Assembly and Disassembly, Cloning, Molecular methods, DNA Helicases genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- 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., (© 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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