2.5 Expression and Purification of the recombinant proteins
The recombinant C. glutamium 13032 strains were first cultivated
into 10 mL BHI liquid medium supplemented with 25 μg/mL chloramphenicol
at 30℃ for overnight, and then 2% inoculation volume were transferred
to 100 mL BHI medium containing 25 μg/mL chloramphenicol at 30°C to an
optical density @600nm of approximately 1 and induced by 1mM IPTG at
30°C for another 20 hours.
The cells were centrifuged (8000 × g, 4℃) for 5 min and resuspended with
10 mL of 50 mM citric acid-sodium dihydrogen phosphate buffer (pH 6.0)
after washing three times. The suspended cells were disrupted by
sonication for 30 min and cells debris was discarded by centrifugation
(8000 × g, 4°C) for 30 min. The soluble supernatant fractions were first
filtered and then further loaded onto a 1 mL Ni affinity column that was
pre-equilibrated with 50 mM wash buffer (20 mM Tris and 500 mM NaCl, pH
7.4), and then elution buffer (20 mM Tris, 500 mM NaCl and 500 mM
imidazole, pH 7.4) was used to elute the unbound proteins and SI with a
linear gradient elution. Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was used for analyzing purified protein. The
concentration of protein was determined through Bradford method.