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Purification of Recombinant Histones and Mononucleosome Assembly

Yiming Zhao,Jingjun Hong

The nucleosome is the basic unit of chromatin. Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a histone octamer . Each histone octamer is composed of two copies each of the histone proteins H2A , H2B , H3 , and H4 . Nucleosomes play an important role in gene expression and regulation. While previous protocols use HPLC (high-performance liquid chromatography) to purify each histone and, after octamer reconstitution, subsequent FPLC (fast protein liquid chromatography) to purify the octamers. Here we present a method to carry out octamer reconstitution and mononucleosome assembly with FPLC only. This basic protocol describes a procedure for histone purification from Escherichia coli (E. coli) , their subsequent reconstitution as octamers, and assembly into mononucleosomes in vitro . Through this protocol, histone octamers and mononucleosomes can be reconstituted on a large scale without the use of HPLC. Overall, this protocol describes an effective new method for the reconstitution of octamers and assembly of mononucleosomes. © 2025 Wiley Periodicals LLC. Basic Protocol : Purification of recombinant histones and mononucleosome assembly