Expression and purification of recombinant yeast Ada2/Ada3/Gcn5 and Piccolo NuA4 histone acetyltransferase complexes

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Author list: Barrios A, Selleck W, Hnatkovich B, Kramer R, Sermwittayawong D, Tan S
Publisher: Elsevier
Publication year: 2007
Volume number: 41
Issue number: 3
Start page: 271
End page: 277
Number of pages: 7
ISSN: 1046-2023
Languages: English-Great Britain (EN-GB)


Abstract

Acetylation of histone tails by histone acetyltransferase (HAT) enzymes is a key post-translational modification of histories associated with transcriptionally active genes. Acetylation of the physiological nucleosome substrate is performed in cells by megadalton complexes such as SAGA and NuA4. To understand how HAT enzymes specifically recognize their nucleosome and not just histone tail substrates, we have identified the catalytic SAGA and NuA4 subcomplexes sufficient to act on nucleosomes. We describe here expression and purification procedures to prepare recombinant yeast Ada2/Ada3/Gen5 subcomplex of SAGA which acetylates histories H3 and H2B on nucleosomes. and the Piccolo NuA4 complex which acetylates histories H4 and H2A on nucleosomes. We demonstrate an unexpected benefit of using the BL21-CodonPlus strain to enhance the purity of metal affinity purified Ada2/Ada3/Gcn5 complex. We also identify Escherichia coli EF-Tu as a contaminant that copurifies with both complexes over multiple chromatographic steps and use of hydrophobic interaction chromatography to remove the contaminant from the Piccolo NuA4 complex. The methods described here will be useful for studies into the molecular mechanism of these enzymes and for preparing the enzymes as reagents to study the interplay of nucleosome acetylation with other chromatin modification and remodeling enzymes. (c) 2006 Elsevier Inc. All rights reserved.


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Last updated on 2019-23-08 at 11:15