2.4 Expression and purification of SuSys
The recombined E. coli was first incubated in 5-mL Luria-Bertani
medium containing 10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract and
50 μg/mL kanamycin, and incubated overnight at 37 °C with continuous
shaking at 200 rpm. Then, 2% (v/v) of the overnight culture was
incubated in shake flasks with 100-mL LB medium containing 50 μg/mL
kanamycin to cultivate for about 2 h at 37 °C.
Isopropyl-β -thiogalactopyranoside in a final concentration of 0.1
mM was added when the culture turbidity (OD600) reached
0.5–0.6, and then the cultivation was continued at 16 °C for another 24
h. The subsequent steps involving purification were performed at 4 °C.
Cells harvested by centrifugation at 5,289 g for 5 min, were
resuspended in an appropriate lysis buffer (500 mM NaCl and 10%
glycerine (v/v) in 20 mM sodium phosphate buffer, pH 8.0) and disrupted
by sonication. After centrifuge twice at 6,665 g for 15 min, the
6 Histidine-tagged proteins in the supernatant were purified by a
high-affinity Ni-charged resin FF prepacked column (GenScript, Nanjing,
China). The recombination proteins were eluted from the column by
stepwise imidazole gradient. Fractions with SuSy activity were pooled
and concentrated in an Amicon® Ultra-15
Centrifugal Filter Unit with an Ultracel-30 membrane (Merck Millipore
Ltd., Ireland), and the buffer was exchanged to 50 mM HEPES-NaOH (pH
7.0). The protein expression and the purity of recombinant enzymes were
analyzed using SDS-PAGE.