To confirm the expression of heterologous enzymes, E. coli BL21(DE3) strains harboring the respective recombinant plasmids were first cultured in 10 mL test tubes containing R/2 medium supplemented with 2 g/L yeast extract, 20 g/L glycerol, and appropriate antibiotics. The cells were grown at 30 °C and were induced with 1 mM IPTG when the OD600 of the culture reached 0.4-0.6. After additional cultivation for 6-10 hr, cells were collected (i.e., 3 mL of cells when their OD600 reached 1.0) by centrifugation at 15,000 g for 1 min at 4 °C.
Cell pellets were washed with 1 mL of cold phosphate-buffered saline (PBS) solution (pH 7.4), centrifuged at 15,000 g for 1 min at 4 °C, and resuspended in 0.3 mL of the same buffer. The resuspended cells were lysed by sonication (High-Intensity Ultrasonic Liquid Processors, Sonics & Materials Inc., Newtown, CT). To precipitate insoluble protein fractions and partially disrupted cells, the sonicated samples were centrifuged at 15,000 g for 10 min at 4 °C. Crude cell lysates were used for total protein expression analysis and the supernatants were used for soluble protein expression analysis.
Cell pellets were washed with 1 mL of cold phosphate-buffered saline (PBS) solution (pH 7.4), centrifuged at 15,000 g for 1 min at 4 °C, and resuspended in 0.3 mL of the same buffer. The resuspended cells were lysed by sonication (High-Intensity Ultrasonic Liquid Processors, Sonics & Materials Inc., Newtown, CT). To precipitate insoluble protein fractions and partially disrupted cells, the sonicated samples were centrifuged at 15,000 g for 10 min at 4 °C. Crude cell lysates were used for total protein expression analysis and the supernatants were used for soluble protein expression analysis.