Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles

Expression and purificationHistonespurification of individual histone proteins and amplification and purification of DNA are the initial steps toward reconstituting nucleosome core particlesNucleosomecore particles. Histone proteins are expressed in E. coli, extracted from inclusion bodies, and purified using ion-exchange chromatography. DNA containing the 147 base pair Widom 601 sequence is amplified in bacteria using a plasmid containing multiple copies of this strong nucleosome positioningNucleosomepositioning sequence. Following alkaline lysis of bacteria, DNA is extracted using phenol and chloroform, released from the vector via restriction enzyme digestionRestriction enzyme digestion, and purified in subsequent precipitation and ion-exchange chromatography steps. Here, we describe a combination of two protocols: one to express and purify recombinantHistonesrecombinant human histones and the other to amplify and purify Widom 601 DNAWidom 601 DNA.