AID/APOBECs are challenging to express and purify due to their genotoxicity to host cells and the proteins’ highly charged surface, which renders them prone to extensive non-specific interactions with DNA/RNA and with other proteins. We have developed unique expertise, methods and reagents that allow for expression and purification of AID/APOBECs and other DNA/RNA-altering enzymes, as well as high-throughput assays to measure every facet of their enzymatic activity. This allows us to tackle biochemical, structural, and molecular aspects of these key enzymes. We measure the enzyme activity parameters (e.g. Michaelis-Menten kinetics) and enzyme: substrate interactions. For substrates, we can use either DNA/RNA substrates ranging in size from simple to complex oligonucleotides, to several kb-long gene sequences and plasmid DNA. To date, we have characterized many of the biochemical features of the AID/APOBECs and discovered that the biochemical properties of these enzymes are a direct and important regulato r of their in vivo activities in immunity and cancer. We establish and optimize multiple expression and purification systems for each DNA/RNA-editing enzyme using different expression hosts (bacterial, yeast, mammalian) and purification strategies (e.g.various fusion tags). For each enzyme, we typically employ multiple expression/purification systems to ensure that our findings reflect a bona fide property. Further, for each enzyme, we construct, express and purify extensive libraries of variants (mutants ,chimeras, species orthologs) which enables in-depth structure: function studies.
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