*2.3. Construction of Lacticaseicin 30 Variant Plasmids for Expression in E. coli Cells*

Each lacticaseicin variant plasmid was generated by site-directed mutagenesis using the pT7-6his-030 plasmid as template and the appropriate primers (Table S2), the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA), and following the recommended instructions.

#### *2.4. Expression and Purification of Lacticaseicin 30 and Its Variants*

Each plasmid constructed in this work was expressed in *E. coli* Rosetta and grown at 37 ◦C in LB broth, supplemented with ampicillin, until reaching the mid-log phase. Expression was then induced by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma Aldrich, St Louis, MO, USA), and the cells were incubated for three additional hours at 37 ◦C with shaking at 160 rpm. Cells were then harvested by centrifugation and resuspended in the Tris–HCl buffer (20 mM Tris–HCl pH 8, 300 mM NaCl). Finally, cells were lysed by sonication (OmniRuptor 4000 Ultrasonic Homogenizer, Omni International, Kennessaw, GA, USA) on ice-cold water and centrifuged at 11,000× *g* for 1 h. The supernatant was loaded onto a nickel resin grafted on a nitrilo-tri-acetic matrix (Protino Ni-NTA Agarose, Machery-Nagel, Düren, Germany) column previously equilibrated with Tris–HCl buffer. The nickel resin was washed with 2 × 10 column volumes of the same buffer supplemented with 30 mM imidazole and the bacteriocins were eluted using 5 column volumes of the previous buffer supplemented with 200 mM imidazole. A desalting step was performed using PD miditrapTM columns (GE Healthcare Life Science, Pollard, UK) to remove imidazole. The histidine-tag was removed with Tev-protease (Sigma Aldrich, St Louis, MO, USA), while the TRX-tag of lacticaseicin 30 peptide variants (Nter-lacticaseicin 30, Cter-lacticaseicin 30 and Nter-H1-lacticaseicin 30) was removed with enterokinase (New England Biolabs, Ipswich, MA, USA). To separate the tag and the peptide without the tag,

additional Ni-NTA chromatography was performed. The purity was verified by Tricine-SDS-PAGE [33]. When necessary, desalted bacteriocin suspensions were lyophilized using the freeze dryer Lyolab 3000 (Thermo Scientific, Waltham, MA, USA) following the recommended instructions. The final concentration of each purified peptide was determined with the bicinchoninic acid assay protein kit (BCA, Sigma Aldrich, St Louis, MO, USA), as recommended by the supplier.
