Abstract
With the identification of increasing number of chromatin modifiers, histone variants, histone post-translational modifications and their cross-talk, it is essential to validate these findings and interactions in vitro for which pure histone complexes are required. Although, the production of such complexes has been described earlier but still it remains a challenge for a non-specialist lab. Here we describe a protocol to quickly obtain large quantities of highly pure histones using bacterial expression system for GST pull-down and reconstitution experiments. In addition, we describe methods to quickly reconstitute and purify H2A/H2B dimers, H3/H4 tetramers and histone octamers for in vitro experiments. We demonstrate that these sub-complexes are properly folded and are hence, true representatives of the actual substrates in vivo. We also show that histones have a propensity to be non-specifically cleaved by proteases. Our results suggest that TEV protease is the most suitable protease while working with histones. The methodology described here should allow researchers to purify histone complexes in three days enabling functional and structural analyses of histone variants, mutants and post-translational modifications.
Highlights
The eukaryotic genome is packaged inside the nucleus of only few microns in diameter [1] with the aid of highlyHow to cite this paper: Bhattacharya, S. and Gupta, S. (2016) A Simple Method to Produce Sub-Nucleosome Complexes of High Purity In Vitro
Apart from the well-established fact that the canonical histones undergo a variety of functionally important post translational modifications (PTM’s), sequence divergent forms of the histones exist which are known as histone variants [3]
We have described a protocol in which histones can be purified and its oligomeric structures reconstituted in very few steps in three days
Summary
The eukaryotic genome is packaged inside the nucleus of only few microns in diameter [1] with the aid of highlyHow to cite this paper: Bhattacharya, S. and Gupta, S. (2016) A Simple Method to Produce Sub-Nucleosome Complexes of High Purity In Vitro. How to cite this paper: Bhattacharya, S. and Gupta, S. (2016) A Simple Method to Produce Sub-Nucleosome Complexes of High Purity In Vitro. The core histones comprise of H2A, H2B, H3 and H4. H2A and H2B form heterodimers and two copies each of H3 and H4 form a tetramer. Two H2A/H2B dimer and one H3/H4 tetramer form the octamer core around which ~147 bp of DNA is wrapped to give rise to nucleosome, which is the fundamental repeating unit of the chromatin [2]. Apart from the well-established fact that the canonical histones undergo a variety of functionally important post translational modifications (PTM’s), sequence divergent forms of the histones exist which are known as histone variants [3]. Novel histone modifiers and binding partners like histone chaperones are being identified with redundant or supposedly non-redundant functions
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