Expression and purification of histone H3 proteins containing multiple sites of lysine acetylation using nonsense suppression.
Lysine acetylation is a common post-translational modification, which is especially prevalent in histone proteins in chromatin. A number of strategies exist for generating histone proteins containing lysine acetylation, but an especially attractive approach is to genetically encode acetyl-lysine residues using nonsense suppression. This strategy has been successfully applied to single sites of histone acetylation. However, because histone acetylation can often occur at multiple sites simultaneously, we were interested in determining whether this approach could be extended. Here we show that we can express histone H3 proteins that incorporate up to four sites of lysine acetylation on the histone tail. Because the amount of expressed multi-acetylated histone is reduced relative to the wild type, a purification strategy involving affinity purification and ion exchange chromatography was optimized. This expression and purification strategy ultimately generates H3 histone uniformly acetylated at the desired position at levels and purity sufficient to assemble histone octamers. Histone octamers containing four sites of lysine acetylation were assembled into mononucleosomes and enzymatic assays confirmed that this acetylation largely blocks further acetylation by the yeast SAGA acetyltransferase complex.
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 1210 Molecular Biology Building, Ames, IA, 50011, USA.
Isaac A
IA
Young
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 1210 Molecular Biology Building, Ames, IA, 50011, USA.
Chitvan
C
Mittal
Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, 1210 Molecular Biology Building, Ames, IA, 50011, USA. Electronic address: knaak@iastate.edu.
Michael A
MA
Shogren-Knaak
D000107:Acetylation; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D018389:Codon, Nonsense / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006657:Histones / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D008239:Lysine / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D029867:Xenopus Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D014982:Xenopus laevis / Q000235:genetics* / Q000378:metabolism
D018389:Codon, Nonsense; D006657:Histones; D029867:Xenopus Proteins; D008239:Lysine
Acetylation; Chromatin; Histone; Nonsense suppression