Heterologous Expression and High Degree Purification of the Restriction Endonuclease SauUSI

Mechanisms that target and destroy foreign nucleic acids are major barriers to horizontal gene transfer (HGT) in prokaryotes. Amongst them, restriction-modification (R-M) systems are found in ≥75% of the sequenced genomes in Bacteria and Archaea. Due to their high target sequence specificity and potent nucleolytic activity, R-M systems are used as a paradigm to elucidate the mechanisms of DNA binding and cleavage. Since these enzymes modulate HGT, they are one of the machineries implicated in the ability of a bacterium to gain antibiotic resistance. This protocol provides a detailed purification strategy for the Type IV restriction endonuclease SauUSI from . This protocol eventually leads to ≥95% purity of protein which can then be used for crystallographic and biochemical purposes. Graphic abstract: Workflow for purification of SauUSI.