Sonication energy input is a critical parameter for preparation of highly active crude cell extracts.
(A) Relative protein synthesis activities from crude extracts prepared with different sonication energy input (in Joules) in 1.5 mL and 0.5 mL cell suspensions. Numbers on the x-axis are total input energy. Fluorescence of sfGFP was measured following a 4 hour CFPS reaction. (B) Protein synthesis activities from crude extracts prepared with different sonication-cooling intervals. For each extract, the same sonication energy input value (556 J) and 1.5 mL cell suspension was used. Energy accumulation was monitored during lysis and then sonication was stopped when the total energy input is reached to 556 J. “C,” positive control, cell extract prepared by 10 L fermentation for culture and homogenization to lyse cells. sfGFP reported was based on the amount of active (fluorescent) sfGFP following a 4 hour CFPS reaction. 95% of the total sfGFP synthesized was active. Values represent averages and error bars represent standard deviation for at least 3 independent experiments.