Optimized Cell Lysis and Extraction Protocol

E. coli BL21 Star™ (DE3) and C495 cells were grown in 2 × YTPG media at 34°C. We grew the strains at 34°C such that we could compare the strains. The MG1655-based C495 strain harbors a temperature sensitive λ-Red recombinase that requires growth at a lower temperature. Antibiotics were not used during cell growth. The cells were grown in a BIOSTAT C-plus fermentor (Sartorious AG, Goettingen, Germany) at 600 rpm for 10 L large-scale cultures. For preparing 1 L scale cultures, cells were grown in 2.5 L baffled tunair shake flasks (IBI Scientific, Peosta, IA) in a 34°C incubator with vigorous shaking at 250 rpm. For smaller scale cultivation (50 to 100 mL culture), 300 mL baffled tunair flasks were used. Unless otherwise specified in the text, the cultured cells were monitored by spectrophotometry (Genesys 10S UV-Vis, Thermo Fisher Scientific, Waltham, MA) until OD₆₀₀ reached at 3.0. The maximum OD₆₀₀ for BL21 Star™ (DE3) and C495 strains measured after 22 h culture that reached saturation were ~10 and ~8, respectively. The cells were harvested in the middle of exponential growth phase by centrifuging at 5000 RCF at 4°C for 15 min and were washed three times with cold Buffer A. Buffer A contained 10 mM Tris-acetate (pH 8.2), 14 mM magnesium acetate, 60 mM potassium glutamate, and 2 mM dithiothreitol. After final wash and centrifugation, the pelleted wet cells were weighed, flash frozen in liquid nitrogen, and stored at −80°C. The thawed cells were suspended in 1 mL of Buffer A per 1 g of wet cell mass. For obtaining standard “control” large-scale cultured cell extract, suspended cells were disrupted by EmulsiFlex-C3 homogenizer (Avestin, Ottawa, Canada) with single pass at a variable pressure of 20,000 to 25,000 psig. In order to remove cell debris and insoluble components in the cell lysate, the lysate was centrifuged twice at 30,000 RCF at 4°C for 30 min. The supernatant was then incubated at 37°C for 60 min with gentle shaking (250 rpm) and centrifuged at 15,000 RCF at 4°C for 15 min. In order to lyse cells by sonication, thawed and suspended cells were transferred into 1.5 mL microtube and placed in an ice-water bath to minimize heat damage during sonication. The cells were lysed using a Q125 Sonicator (Qsonica, Newtown, CT) with 3.175 mm diameter probe at frequency of 20 kHz and 50% of amplitude. The input energy (Joules) was monitored and recorded during sonication. The lysate was then centrifuged once at 12,000 RCF at 4°C for 10 min. The run-off reaction (37°C at 250 rpm) and second centrifugation (10,000 RCF at 4°C for 10 min) were performed for strain C495. However, in the case of strain BL21 Star™ (DE3), the run-off reaction and second centrifugation step were not needed. The total amount of protein in cell extract was quantified by Bradford assay. All extracts contained 40.7 ± 2.6 mg/mL of total E. coli proteins. All of prepared cell extract was flash frozen in liquid nitrogen and stored at −80°C until use.