Evolving Chromodomain Reader Domains for Non-Natural Histone Mark Recognition

The development of new methods to bind and enrich histones that contain specific, non-natural histone PTMs has broad reaching implications to better understand many crucial biological processes, such as replication, transcription, and mitosis. To evolve a chromodomain reader protein, a Cbx1 gene III phage display strategy was developed and characterized in a phage ELISA system to replicate previously characterized binding data of a suite of Cbx1 binding probes ranging from Kd values of 320 µM to 130 nM. To accurately characterize these binding dynamics, it was necessary to incorporate at least 1/10th the amount of biotin to the amount of biotinylated peptide. Four parallel phage display selections were completed to attempt to evolve stronger binders to H3K9me3 and H3K9me3S10p from a naïve library of five amino acid randomized NNK codons in the EPEEN region rationally targeted by structural and computational information. The selection process showed greater than ten-fold enrichment for selections to the different histone tail peptides, and NGS data showed that the WT Cbx1 sequence was highly enriched in both libraries, and an additional RAWMI mutant was moderately enriched in the H3K9me3S10p selection and not as strongly selected against as other amino acids in the H3K9me3 selection. The Cbx1 RAWMI mutant protein has not shown any measurable binding via ITC to either of the H3 PTM peptides. This study sets the stage for future evolution of reader domains for improved function as a biological enrichment tool.Bachelor of Scienc