Expression, purification and proteomic analysis of recombinant histone H4 acetylated at lysine 16

• Authors: Satish Kallappagoudar, Emory University; Eric B Dammer, Emory University; Duc Duong, Emory University; Nicholas Seyfried, Emory University; John Lucchesi, Emory University
• Journal Title: Proteomics
• Volume: Volume 13, Number 0
• Publisher: Wiley-VCH Verlag | 2013-05, Pages 1687-1691
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1002/pmic.201300025
• Subjects: Biology, Molecular; Biology, Neuroscience
• Keywords: Gene expression; Histone; Nucleosome; Technology

Abstract

Many histone co-valent modifications have been identified and shown to play key regulatory roles in eukaryotic transcription, DNA damage repair and replication. In vitro experiments designed to understand the mechanistic role of individual modifications require the availability of substantial quantities of pure histones, homogeneously modified at specific residues. We have applied the amber stop codon/suppressor tRNA strategy to the production of histone H4 acetylated at lysine 16, a particularly important isoform of this histone. Our success relies on adapting the H4 DNA sequence to the codon preference of E. coli and on preventing the premature decay of the H4 mRNA. These modifications to the original procedure render it easily applicable to the generation of any co-valently modified histone H4 isoform.