Bl21 codonplus de3 ripl chemically competent cells

BL21-CodonPlus (DE3)-RIPL chemically competent cells (Agilent) were transformed with pET-24a-mΔN-PDE3B and plated on LB-Agar plates containing 25μg/mL kanamycin and 25μg/mL chloramphenicol. Expression cultures were inoculated at 1:20 dilution from saturated overnight cultures and grown with 25μg/mL kanamycin and 25μg/mL chloramphenicol at 37 ºC to OD₆₀₀~1.0. Protein expression was induced with 1 mM IPTG at 37 ºC for 3 h. The cultures were centrifuged at 4 ºC and 4000 × g for 5 min, yielding ~4 g/L cell paste, which was frozen in liquid N₂.
Inclusion bodies containing ΔN-PDE3B were purified similarly to a published protocol^{55 (link)}. The cell paste was suspended in 5 mL/g of 50 mM sodium phosphate, 10% glycerol, 1 mM PMSF, pH 7.6, and lysed by passage once through a French pressure cell at 14000 psi. The lysate was incubated at 25 ºC with 5 U/g DNase for 15 min and centrifuged at 15000 × g for 20 min at 4 °C. The pellet was resuspended in 5 mL/g 100 mM Tris, 4% (v/v) Triton X-100, 2 M urea, pH 8.0, and centrifuged at 15,000 × g for 20 min. The resulting pellet was resuspended and centrifuged once more. Finally, the pellet was washed twice via resuspension and centrifugation in 5 mL/g water. (Addition of 10% Buffer B improved pelleting of the inclusion bodies.) The isolation yielded ~0.1 g inclusion bodies/g of cell paste, which were stored at −20ºC.