Faculty Collaboration Database

Preparation of Recombinant Histones and Widom 601 DNA for Reconstitution of Nucleosome Core Particles. Methods Mol Biol 2023;2599:163-175

Date

11/26/2022

Pubmed ID

36427149

DOI

10.1007/978-1-0716-2847-8_12

Scopus ID

2-s2.0-85142940771 (requires institutional sign-in at Scopus site)   3 Citations

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 EA

Author

Emma A. Morrison PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

DNA
DNA Restriction Enzymes
Escherichia coli
Histones
Humans
Nucleosomes