Publication protocol
Recombinant plasmids were first screened for the TRP36-fusion protein expression using L- (+)-arabinose as the inductor. After a small-scale experiment to determine the optimal concentration of the sugar, one positive clone was grown overnight in LB medium containing 100 μg/mL ampicillin at 37 °C under shaking (250 rpm). This overnight culture was inoculated into 500 mL of LB medium in a 1 L culture flask for the large-scale expression experiment. The culture was maintained at 37 °C under shaking until an OD550 of 0.5. L-arabinose was then added to a final concentration of 0.002%, and the culture was grown for 4 h at 25 ºC under shaking. After reaching the corresponding OD, cells were harvested by centrifugation (9,500 x g) for 20 min at 4 ºC. The pellet was suspended in phosphate buffer saline (PBS) at pH 7.4, and a 100 μL sample was analyzed by 12% SDS-PAGE under denaturing and reducing conditions, as described by Laemmli (1970). The remainder of the preparation was centrifuged, and the pellet was resuspended in 10 mL of ice-cold binding buffer (20 mM sodium phosphate buffer, pH 7.8, containing 500 mM NaCl and 10 mM imidazole) plus lysozyme (1 mg/ mL), mixed with microglass beads (0.1 mm) v/v, and subjected to three 10 s pulses at 10 s intervals in the Bead Beater (model 1107900, Biospec, Bartlesville, USA). After glass bead sedimentation, the supernatant was transferred to a Falcon tube to which DNase and RNase were added to a final concentration of 5 and 10 μg/mL, respectively. The mixture was centrifuged to pellet the debris and the microbeads, and the supernatant was stored for the future isolation of the TRP36-fusion protein.
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Manufacturer protocol
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