RGP1-pET55dest

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	E. coli Origami 2™ cells transformed with the RGP1-pET55dest plasmid were grown at 37°C in autoinduction medium for 5 h. Then the culture was cooled to 18°C for protein expression and grown for 24 h.

Protocol tips
E. coli Origami 2™ cells transformed with the RGP1-pET55dest plasmid were grown at 37°C in autoinduction medium for 5 h. Then the culture was cooled to 18°C for protein expression and grown for 24 h.

Publication protocol

E. coli Origami 2™ cells transformed with the RGP1-pET55dest plasmid were grown at 37°C in autoinduction medium for 5 h. Then the culture was cooled to 18°C for protein expression and grown for 24 h. Cells were harvested by centrifugation at 5000 x g for 10 min at 4°C and the cell pellet was frozen and stored at −80°C. The cell pellet was thawed and resuspended in 30 mL 10 mM Tris pH 8.5, 100 mM NaCl, 1 mM EDTA at 4°C. The resuspension was divided into 31 1 mL portions in 1.5 mL microcentrifuge tubes and again pelleted by centrifugation. This step serves to wash the cells and to apportion them for treatment with the different lysis buffers, and is crucial for reproducibility [21]. Then, the pellets were resuspended in 1 mL of each lysis buffer. 0.15 mg/mL lysozyme and 0.12 mg/mL protease inhibitor (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF)) were added to each sample and the resuspensions were incubated on ice for 5 min. After that, the cells were disrupted by sonication, and the lysate was centrifuged for 10 min. at 16,000 x g at 4°C. Equal volumes of each supernatant were analyzed on SDS-PAGE and the level of protein recovery in each condition was assessed by visually comparing the intensity of the bands on a western blot against a cloning scar (see next section).

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