E. coli BL21(DE3) cells were transformed with the
constructed plasmid to express the enzyme. The experimental procedure
for gene expression and soluble protein analysis is described inFigure S1 . A glycerol stock for inoculation was
prepared using a Luria-Bertani (LB) medium containing 50 μg/mL
kanamycin. Glycerol stocks (30 μL) were inoculated into 3 mL
of LB-Autoinduction medium (Overnight Express Autoinduction System1,
Merck) containing 50 μg/mL kanamycin and cultivated at 30 °C
with shaking at 250 rpm. The bacteria were grown to an OD600 of ∼0.6
to 0.8. Protein expression was induced at 15 °C for 24 h or at
30 °C for 12 h, with shaking at 250 rpm. After induction, 2 mL
of the cultures was centrifuged at 10 000g for 5 min at 4 °C. The cell pellets were resuspended in 500
μL of 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH
6.5). Whole-cell lysates were prepared by disrupting the cell suspension
via ultrasonication for 10 min on ice using a Bioruptor UCD-250 (TOSHO
DENKI, Japan). Soluble and insoluble fractions were prepared from
the whole-cell lysate by centrifugation at 20 000g for 20 min at 4 °C. The supernatant was collected as the soluble
fraction, and the precipitate was resuspended in the same volume of
MES buffer as the supernatant (insoluble fraction). Each fraction
(2.5 μL) was analyzed by SDS-PAGE using a 10% (w/v) polyacrylamide
gel.
constructed plasmid to express the enzyme. The experimental procedure
for gene expression and soluble protein analysis is described in
prepared using a Luria-Bertani (LB) medium containing 50 μg/mL
kanamycin. Glycerol stocks (30 μL) were inoculated into 3 mL
of LB-Autoinduction medium (Overnight Express Autoinduction System1,
Merck) containing 50 μg/mL kanamycin and cultivated at 30 °C
with shaking at 250 rpm. The bacteria were grown to an OD600 of ∼0.6
to 0.8. Protein expression was induced at 15 °C for 24 h or at
30 °C for 12 h, with shaking at 250 rpm. After induction, 2 mL
of the cultures was centrifuged at 10 000g for 5 min at 4 °C. The cell pellets were resuspended in 500
μL of 100 mM 2-morpholinoethanesulfonic acid (MES) buffer (pH
6.5). Whole-cell lysates were prepared by disrupting the cell suspension
via ultrasonication for 10 min on ice using a Bioruptor UCD-250 (TOSHO
DENKI, Japan). Soluble and insoluble fractions were prepared from
the whole-cell lysate by centrifugation at 20 000g for 20 min at 4 °C. The supernatant was collected as the soluble
fraction, and the precipitate was resuspended in the same volume of
MES buffer as the supernatant (insoluble fraction). Each fraction
(2.5 μL) was analyzed by SDS-PAGE using a 10% (w/v) polyacrylamide
gel.