2.3 Cell disruption and purification:
The cell pellets were thawed on ice and resuspended in lysis buffer (50
mM phosphate buffer containing 300 mM NaCl and 10 mM imidazole, pH 8.0),
with use of 10 mL buffer per 5 g of wet cells. Then the cells were
disrupted by sonication (2 x 4 min, 50% duty cycle; Sonicator QSonica
Q500 Ultra Sonicator) with cooling on ice. Cell debris was removed by
centrifugation at 18000g for 30 min at 4 °C. The supernatant was
filtered through a 0.25 μm PVDF filter and was loaded onto a Ni-NTA
column (Thermo scientific) equilibrated with lysis buffer. After loading
of the filtered lysate, the column was washed with six column volumes of
wash buffer (50 mM phosphate buffer containing 300 mM NaCl and 20 mM
imidazole, pH 8.0). Finally, the protein was eluted with elution buffer
(50 mM phosphate buffer containing 300 mM NaCl and 250 mM imidazole),
and fractions containing target enzyme (determined by SDS-PAGE) were
pooled and dialyzed at 4 °C against Tris-HCl buffer (20 mm, pH 7.5).
Aliquots of concentrated enzymes with approximately 10-20 μM,
flash-frozen on powdered dry ice, and stored at -80 °C until later use.
Protein expression and purity was assessed by SDS-PAGE (Figure S3).