Luna c5 column

Plasmids were constructed and used to transform E. coli using a previously reported method.^{30 (link)} Single colonies were selected and amplified
in 5 mL of starter cultures in LB containing 50 μg/mL of kanamycin
overnight. The starter culture was introduced into 1 L of LB medium
containing 50 μg/mL of kanamycin and incubated at 37 °C
with continuous shaking at 220 rpm until it reached an OD₆₀₀ of approximately 0.8. Subsequently, the cultures were cooled on
ice for approximately 15 min before the addition of isopropylthio-β-galactoside
(IPTG) to a final concentration of 0.5 mM. Following this, the cultures
were shaken overnight at 180 rpm at 18 °C. Cells were harvested
by centrifugation at 6000g for 15 min at 4 °C.
The supernatant was discarded, and the pellet was stored at −80
°C before purification.
The purification method was identical
to that described for mCylL_L, followed by an Agilent preparative
HPLC step using a Phenomenex Luna C5 column (5 μm, 250 ×
10 mm²). The following gradient conditions were used for
the purification process: flow rate of 4 mL/min, solvent A H₂O + 0.1% TFA, solvent B MeCN + 0.1% TFA; 0–5 min with 2% B,
5–35 min using a gradient of 2–80% B, and 35–40
min using a gradient of 80–100% B. mBuvA eluted at 27–32
min, corresponding to 62–72% B.

Nisin was obtained by extraction from a Sigma-Aldrich
product that contains about 5% nisin from Lactococcus
lactis. The process involved suspending 100 mg of
the commercial product in 10 mL of extraction solvent consisting of
80% acetonitrile (MeCN)/20% H₂O + 0.1% trifluoroacetic
acid (TFA) in a 50 mL conical tube, followed by vigorous vortexing.
After centrifuging at 4000g for 10 min, the supernatant
was collected, and the extraction of the insoluble material was repeated
twice with 10 mL of the same solvent. The combined supernatant was
then filtered using a 10 K Amicon Ultra-15 Centrifugal Filter,
and the resulting flowthrough was collected. The solution was frozen
with liquid nitrogen and lyophilized. The resulting dry solid was
redissolved in 10% MeCN/90% H₂O + 0.1% TFA, filtered, and
injected onto an Agilent preparative HPLC system for purification
using a Phenomenex Luna C5 column (10 μm, 250 × 10 mm²). The following gradient conditions were used for the purification
process: flow rate of 4 mL/min; solvent A H₂O + 0.1% TFA,
solvent B MeCN + 0.1% TFA; 0–5 min with 2% B, 5–15 min
using a gradient of 2–40% B, and 15–30 min using a gradient
of 40–100% B, with nisin eluting at approximately 20–22
min, corresponding to around 40% solvent B.