Genetically encoding lysine modifications on histone H4

Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.