Abstract
Functional and structural studies of histone-chaperone complexes, nucleosome modifications, their interactions with remodelers and regulatory proteins rely on obtaining recombinant histones from bacteria. In the present study, we show that co-expression of Xenopus laevis histone pairs leads to production of soluble H2AH2B heterodimer and (H3H4)2 heterotetramer. The soluble histone complexes are purified by simple chromatographic techniques. Obtained H2AH2B dimer and H3H4 tetramer are proficient in histone chaperone binding and histone octamer and nucleosome formation. Our optimized protocol enables rapid purification of multiple soluble histone variants with a remarkable high yield and simplifies histone octamer preparation. We expect that this simple approach will contribute to the histone chaperone and chromatin research.
Highlights
T HE genome of eukaryotic cells has to be packed within a small nucleus, but at the same time accessible to different macromolecular machineries required for DNA replication, transcription, recombination, damage and repair.[1]
We reasoned that simultaneous expression of all four histones in Escherichia coli could put a burden on the protein synthesis, lowering the amounts of the purified complex
Individual histones expressed in bacteria are insoluble and form inclusion bodies
Summary
T HE genome of eukaryotic cells has to be packed within a small nucleus, but at the same time accessible to different macromolecular machineries required for DNA replication, transcription, recombination, damage and repair.[1]. The most straightforward modification of the original protocol is "one pot refolding" method.[14] In this method all four X. laevis histones are expressed individually into inclusion bodies, mixed together and denatured in guanidine hydrochloride After this solubilisation step, histone octamer is reconstituted by dialysis against sodium chloride and further purified using affinity and size exclusion chromatography. For structural studies histone proteins from X. laevis and H. sapiens are most commonly used These studies include deciphering the interactions within NCP itself or with its binding proteins,[18] as well as studying histone-chaperone mechanisms of recognition.[19] Structural studies require large amounts of highly purified components. The N-terminal 6xHis-tag was kept at the H2A N-terminus and at the H4 C-terminus, as in the original plasmid
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