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 mCylLL, 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.
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 mCylLL, 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.
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