pET28a-FTP

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	Following cloning and chemical synthesis, the FTP gene was subcloned into pET28a and the vector transformed into E. coli BL21 (DE3). Cells were then inoculated on LB plates supplemented with kanamycin (50 μg/ml), followed by incubation overnight at 37°C.

Protocol tips
Following cloning and chemical synthesis, the FTP gene was subcloned into pET28a and the vector transformed into E. coli BL21 (DE3). Cells were then inoculated on LB plates supplemented with kanamycin (50 μg/ml), followed by incubation overnight at 37°C.

Publication protocol

"Following cloning and chemical synthesis, the FTP gene was subcloned into pET28a and the vector transformed into E. coli BL21 (DE3). Cells were then inoculated on LB plates supplemented with kanamycin (50 μg/ml), followed by incubation overnight at 37°C.

Positive clones were confirmed using colony PCR. One was used to obtain a 5-ml pre-inoculate of LB medium, which in turn was used to inoculate (1:100) 100 ml LB medium for culturing at 37°C until an OD600 nm of 0.4–0.6 was reached. Aliquots (10 ml) were collected, increasing amounts of IPTG added (0.01, 0.1, 0.3, 0.5 and 1.0 mM) and each culture was incubated at different temperatures (20, 25, 30 or 37°C), under shaking speeds of 200 rpm for 4 or 8 h. Cells were pelleted at 8000×g for 15 min at 4°C, the supernatant discarded and the cells resuspended in lysis buffer (20 mM Tris/HCl, 200 mM NaCl, 200 μg/ml lysozyme and 1 mM PMSF, protease inhibitor). After 15 min, 1% Triton X-100 was added and the homogenates submitted to five cycles of freezing-thawing before centrifugation to separate soluble and insoluble protein fractions. Both parts were quantified (Bradford assay) and analysed by SDS/PAGE. However, as the pET28a vector expressed mainly insoluble FTP, we also cloned the FTP gene into the pET SUMO vector (Invitrogen) and essentially repeated the above studies in E. coli B21 cells, except that two shaking speeds were used (130 and 200 rpm) and incubation times increased (12, 16 or 21 h)."

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