title: Expression, purification and proteomic analysis of recombinant histone H4 acetylated at lysine 16.
abstract: Many histone covalent 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 covalently modified histone H4 isoform.
authors.affiliation: Department of Biology, Emory University, Atlanta, GA 30322, USA.
authors.forename: Satish
authors.initials: S
authors.lastname: Kallappagoudar
authors.forename: Eric B
authors.initials: EB
authors.lastname: Dammer
authors.forename: Duc Minh
authors.initials: DM
authors.lastname: Duong
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Seyfried
authors.forename: John C
authors.initials: JC
authors.lastname: Lucchesi
mesh_terms: D000107:Acetylation; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018388:Codon, Terminator; D004926:Escherichia coli; D006657:Histones / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D053719:Tandem Mass Spectrometry
chemical_list: D018388:Codon, Terminator; D006657:Histones; D011994:Recombinant Proteins; D008239:Lysine
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