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Streamlined extract preparation for Escherichia coli-based cell-free protein synthesis by sonication or bead vortex mixing
 
Prashanta Shrestha, Troy Michael Holland, and Bradley Charles Bundy
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Supplementary Material





Materials and methods

Shake flasks cell culture

Cell growth for extract preparation was performed using BL21 Star (DE3) cells harboring the pEVOL-pPrF plasmid (39) in 2.5-L baffled tunair flasks (IBI Scientific, Peosta, IA, USA). Cells were cultured at 37°C with 280 rpm agitation in a rotary incubator shaker (value in g not specified for all incidences of rpm). The fermentation was performed with and without the presence of 100 mM 3-morpholinopropanesulfonic acid (MOPS) in 2× yeast extract and tryptone (YT) growth media. The fermentations were inoculated with 1 mM isopropyl β-D-1-thiogalactopyranoside and 0.02% (w/v) L-arabinose at an optical density (OD600) of 0.6. Cells were harvested at mid to late logarithmic growth phase at an OD600 of 2.7 to 3.8 4 h after induction by centrifugation at 8000 rpm at 4°C for 30 min. Cells were then washed by suspending in 10 mL ice-cold buffer A (10 mM Tris base, 14 mM magnesium acetate, 60 mM potassium glutamate, and 1 mM dithiothreitol) per gram of cell and centrifuged at 6000 rpm at 4°C for 30 min and subsequently resuspended in 1 mL ice-cold buffer A per gram of cell in preparation for cell lysis. Finally, the cell suspension was flash-frozen in liquid nitrogen and stored at -80°C prior to lysis. Note that the pEVOL-pPrF plasmid and its induction with arabinose are not necessary for extract preparation. However, its inclusion in the E. coli cells used for extract preparation enables the option of cell-free production of proteins containing unnatural amino acids.

Cell lysis and extract preparation

High-pressure homogenization. Thawed cell suspensions were lysed with three passes through an Emulsiflex-B15 French press-style high-pressure homogenizer (Avestin, Ottawa, ON, Canada) at 24,000 psi with sample cooling for 1 min in an ice-water bath after the second pass. The lysate was centrifuged at 12,000× g for 10 min at 4°C, and the pellet was discarded. The supernatant was carried forward for a run-off reaction by incubating at 37°C with 280 rpm agitation for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Sonication. Thawed cell suspensions were lysed using a Vibra-cell VCX 400 probe sonicator with a CV 26 probe (tip diameter of 3 mm; Sonics & Materials, Newtown, CT, USA) at a frequency of 20 kHz. The sample vial was kept in an ice-water bath to prevent significant heating in the sample during sonication. The lysate was centrifuged for 30 min at 12,000× g at 4°C, and the run-off reaction was performed by incubating the supernatant at 37°C with 280 rpm agitation for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use. Sonication was performed by cycling at the sonication and cooling intervals as shown in Table 1.





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Bead vortex mixing. Glass beads of 0.1 mm diameter (Scientific Industries, Bohemia, NY, USA) at 10%, 20%, 50% or 80% (w/v) beads-to-cell suspension ratio were used for cell lysis in 1.5- or 2-mL microcentrifuge tubes. The cell and bead suspension was vortex mixed on the table top vortex mixer, Fisher Vortex Genie 2 (Scientific Industries), at 3200 rpm for different time intervals with a 1-min cooling period between each vortex mixing. The lysate was centrifuged twice at 12,000× g at 4°C for 30 min with the supernatant retained each time. The run-off reaction was performed at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Lysozyme incubation. Hen egg white lysozyme (Sigma-Aldrich, St. Louis, MO, USA) was added to the thawed cell suspension at a concentration of 0.1 mg/mL and incubated at 37°C with gentle shaking (80 rpm) for 3 h, similar to as reported previously (40). The lysate was centrifuged at 12,000× g at 4°C for 10 min. The run-off reaction was performed by incubating the supernatant at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

Freeze-thaw cycling. Thawed cell suspensions of 500 µL in 1.5-mL microcentrifuge tubes were subjected to three freeze-thaw cycles by freezing the cell suspensions on liquid nitrogen or dry ice for 15 min and thawing for 15 min in a water bath at 25°C, similar to previously reported methods (41, 42). The lysate was centrifuged at 12,000× g at 4°C for 10 min. The run-off reaction was performed by incubating the supernatant at 37°C and 280 rpm shaking for 30 min. The extract was flash-frozen in liquid nitrogen and stored at -80°C until use.

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