pET-21b(+)/Pro j 1

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	The constructs were transformed into competent E. coli BL21 (DE3) cells (Novagen). For production of rPro j 1, the recombinant plasmid pET-21b(+)/Pro j 1 was inoculated into 3 ml of lysogeny broth (LB) medium containing 100 μg/ml of ampicillin and incubated at 37°C. Expression was induced by isopropyl β-D thiogalactopyranoside (IPTG) (0.6 mM) and then the cells were harvested by centrifugation (3,500 ×g, 15 min, 4°C) and resuspended in lysis buffer.

Protocol tips

The constructs were transformed into competent E. coli BL21 (DE3) cells (Novagen). For production of rPro j 1, the recombinant plasmid pET-21b(+)/Pro j 1 was inoculated into 3 ml of lysogeny broth (LB) medium containing 100 μg/ml of ampicillin and incubated at 37°C. Expression was induced by isopropyl β-D thiogalactopyranoside (IPTG) (0.6 mM) and then the cells were harvested by centrifugation (3,500 ×g, 15 min, 4°C) and resuspended in lysis buffer.

Publication protocol

"The constructs were transformed into competent E. coli BL21 (DE3) cells (Novagen). For production of rPro j 1, the recombinant plasmid pET-21b(+)/Pro j 1 was inoculated into 3 ml of lysogeny broth (LB) medium containing 100 μg/ml of ampicillin and incubated at 37°C. Expression was induced by isopropyl β-D thiogalactopyranoside (IPTG) (0.6 mM) and then the cells were harvested by centrifugation (3,500 ×g, 15 min, 4°C) and resuspended in lysis buffer (50 mM Tris–HCl pH 6.8, 15 mM imidazole, 100 mM NaCl, 10% glycerol, and 0.5% Triton X-100). Finally, the cells were disrupted by subjecting them to three freeze-thaw cycles in liquid nitrogen. Purification of rPro j 1 was performed with Ni-NTA affinity chromatography (Invitrogen) from the soluble phase of lysate, following the manufacturer’s instructions.
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