Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles. Methods Mol Biol 2023;2599:163-175
Date
11/26/2022Pubmed ID
36427149DOI
10.1007/978-1-0716-2847-8_12Scopus ID
2-s2.0-85142940771 (requires institutional sign-in at Scopus site)Abstract
Expression and purification of individual histone proteins and amplification and purification of DNA are the initial steps toward reconstituting nucleosome core 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 positioning sequence. Following alkaline lysis of bacteria, DNA is extracted using phenol and chloroform, released from the vector via restriction 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 recombinant human histones and the other to amplify and purify Widom 601 DNA.
Author List
Paintsil EA, Morrison EAAuthor
Emma A. Morrison PhD Assistant Professor in the Biochemistry department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
DNADNA Restriction Enzymes
Escherichia coli
Histones
Humans
Nucleosomes
