pQEhCP1

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	The recombinant EhCP1 protein was expressed in the cytosolic compartment of SHuffle Express cells harboring the pQEhCP1 plasmid. A fresh culture of transformed cells was sub-cultured (1:100) and grown at 37 °C for 2 h with shaking (300 rpm). Gene expression was induced by addition of IPTG to a final concentration of 0.5 mM.

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The recombinant EhCP1 protein was expressed in the cytosolic compartment of SHuffle Express cells harboring the pQEhCP1 plasmid. A fresh culture of transformed cells was sub-cultured (1:100) and grown at 37 °C for 2 h with shaking (300 rpm). Gene expression was induced by addition of IPTG to a final concentration of 0.5 mM.

Publication protocol

"The recombinant EhCP1 protein was expressed in the cytosolic compartment of SHuffle Express cells harboring the pQEhCP1 plasmid. A fresh culture of transformed cells was sub-cultured (1:100) and grown at 37 °C for 2 h with shaking (300 rpm). Gene expression was induced by addition of IPTG to a final concentration of 0.5 mM. Expression was allowed by further growing at 30 °C for 18 h with shaking. Bacterial cells were harvested by centrifugation at 9300 x g, 10 °C for 10 min. Five cell pellets, each from a 200-mL batch, were obtained and preserved at − 20 °C.

Bacterial protein extracts were prepared by cell lysis under native conditions. Each pellet was suspended in 5 mL of TT-L buffer (1% Triton X-100; 100 mM Tris-HCl, pH 8.0; supplemented with 0.2 mg/mL lysozyme) and thoroughly mixed by rocking for 5 min. Cell lysis was achieved by sonication, using a typical procedure (10 cycles: 30 s ON, 30 s OFF; on ice bath), followed by rocking for 10 min. The cellular debris was removed by centrifugation at 9300 x g for 15 min at 10 °C. The supernatant (soluble fraction) was further cleared by centrifugation at 16000 x g, 10 °C for 15 min. Protein concentration was determined by conducting a Bradford assay [39], using BSA as the standard. Protease activity was estimated by conducting a chromogenic assay [40], using Z-Arg-Arg-pNA as the substrate."

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