Rp hplc

Peptides were synthesized using an automated peptide synthesizer (Biotage, Uppsala, Sweden) in a 0.1 mmol scale by standard protocols for Fmoc solid‐phase synthesis. Rink amide ChemMatrix resin (Biotage) was used as solid phase to obtain C‐terminally amidated peptides. Stearic acid (Sigma‐Aldrich, Munich, Germany) and PEG (PEG1000, polydisperse—Jenkem, Plano, TX; PEG600, monodisperse—Chempep, Wellington, FL) were coupled like standard amino acids, with coupling times of 18 hours and 24 hours, respectively. For the generation of intramolecular disulfide bonds, the peptidyl resin was treated with 1.6 eqv of thallium (III) trifluoroacetate (Sigma‐Aldrich) in DMF (Sigma‐Aldrich) for 30 minutes.21 Cleavage from resin was done following standard protocol (95% trifluoroacetic acid [TFA] [Sigma‐Aldrich], 2.5% TIS [Sigma‐Aldrich], 2.5% MQ water). The peptides were purified by RP‐HPLC (Agilent, Santa Clara, CA) using a C3 column and 20%‐90% acetonitrile gradient in water (0.1% TFA). Molecular weights of the peptides were analyzed by MALDI‐TOF mass‐spectrometry, and purities were determined by UPLC (Waters, Milford, MA). Disulfide bond formation was confirmed with Ellmann's test (Ellmann's reagent, Sigma‐Aldrich).

Plantaricin YKX was purified by protein precipitation, SP-Sepharose cation-exchange chromatography (80 × 2.0 cm, Sigma, Santa Clara, CA, United States), and reversed-phase high-performance liquid chromatography (RP-HPLC) (Agilent Technologies, Palo Alto, CA, United States). First, L. plantarum cells (50 mL) were cultured in MRS (1 L) at 30°C for 24 h. Next, the cell-free supernatants were concentrated to 1/5th of the initial volume using a rotavapor (RV-8V, IKA, Staufen, Germany). After that, ammonium sulfate was added to 50% (v/v) saturation and stirred overnight at 4°C. The obtained residue was suspended in 20 mM disodium hydrogen phosphate-citric acid buffer (pH 5.0), loaded on the SP-Sepharose column, and eluted with the same buffer at 0.5 mL/min. Next, the collected fraction showing the highest activity was scanned to obtain the maximal absorption wavelength and then precipitated with 50% aqueous methanol (v/v) and vortexed for 1 min. Further, it was fractionated using a Dionex UltiMate 3000 HPLC system with a photodiode array detector coupled with an Agilent HC-C18 column (5 μm, 250 mm × 4.6 mm). A linear gradient of ACN/water (10–95%) over 40 min was used for elution at 0.5 mL/min. The antibacterial activity was determined by agar well diffusion assay using A. acidoterrestris DSM3922 as an indicator. The Bradford assay measured the protein concentration.

Protein molecular weight and purity were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the used acrylamide and separation gel was 5% and 16%, respectively [54 (link),55 (link)]. The molecular weight of the protein was determined in comparison to the Unstained Protein 26610 molecular marker kit (Thermo Fisher Scientific, Madison, CT, USA). The protein was visualized by staining the gel with Coomassie Brilliant Blue-250 (Thermo Fisher Scientific, Madison, CT, USA).
Reversed-phase high-performance liquid chromatography (RP-HPLC) is featured by its high resolution and separation efficiency. The protein purity was further confirmed by using RP-HPLC (Agilent, Foster City, CA, USA). The protein was loaded onto 5 μm ZORBAX® 300SB-C8 column (4.6 × 150 mm, Agilent, Foster City, CA, USA) linked to a series 1100 HPLC system which was pre-equilibrated with 0.1% (v/v) TFA for 2 min. In the solvent, there were solvent A and solvent B, which were 0.1% (v/v) TFA in Milli-Q water and 0.1% (v/v) TFA in acetonitrile, respectively. The flow rate was set to 1 mL/min, and the linear gradient process for solvent A (40–90%) and B (90–100%) was 30 min and 10 min, respectively. Then, the protein was kept in 100% solvent B for 5 min. The detection wavelength was 280 nm and the column temperature was 30 °C [56 (link),57 (link)].