TITLE:
A Simple Method to Produce Sub-Nucleosome Complexes of High Purity In Vitro
AUTHORS:
Saikat Bhattacharya, Sanjay Gupta
KEYWORDS:
Histone, Nucleosome, H2A/H2B Dimer, Histidine-Tag, Recombinant
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.7 No.3,
March
8,
2016
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.