Next, ncAA-modified peptides were purified via IMAC, utilizing the N-terminally His-tagged nisin leader. For both the WT nisin as control and the SCS samples, antimicrobial activity was detected from elution fractions and concentration of bioactive nisin was evident as judged from the size of inhibition halos (Supplementary Figure S3A). Consequently, IMAC purification yielded samples of improved purity and antimicrobial activity. LC–MS analysis was conducted to verify incorporation of the ncAA into the RiPP. Likewise, the WT construct without an in-frame amber stop codon was recombinantly produced, purified, and analyzed. As anticipated, purified peptide fractions were found to contain NisBC-processed [i.e., (Me)Lan-containing, cyclized] nisin, for the SCS samples carrying BocK at positions 4 and 12, respectively (see Figure 2B and Supplementary Figure S3B for theoretical and observed molecular masses).