2.5 Production, purification and analysis of recombinant SaCas9 (rSaCas9)
The coupled in vitro transcription/translation reaction for the production of rSaCas9 was performed essentially as described [12]. Briefly, 150 µl of tobacco BY-2 cell-free lysate (BYL, provided by M. Buntru, Fraunhofer IME) was placed in individual wells in a 96-well plate. A total volume of 8 ml of BYL was used. Each well was supplemented with 6 µg of template DNA (pIVEX_SaCas9). A vector coding for enhanced yellow fluorescent protein (eYFP) was used as a positive control. Empty wells were filled with distilled water to prevent evaporation by increasing the humidity, and the plate was sealed with a lid. The plate was incubated on an orbital shaker (Lab-Therm LT-X, Adolf Kühner AG, Birsfelden, Switzerland) at 500 rpm, 25 °C and a humidity of 72% for 48 hours (a sample was taken at 24 h to monitor protein formation).
After completion of the in vitro transcription/translation reaction, the cell-free reaction mixtures containing rSaCas9 were removed from the wells, combined, and supplemented with 10x washing buffer (Strep-Tactin XT kit, IBA Lifesciences GmbH, Göttingen, Germany) at a ratio of 9+1. Insoluble material was removed by centrifugation at 16,100 ×g for 20 minutes at 4 °C. The supernatant was loaded onto a gravity flow column containing a 1 ml bed volume of Strep-Tactin XT superflow high capacity matrix (IBA) equilibrated with washing buffer. After loading, the matrix was washed with 5 volumes of washing buffer, and protein was eluted subsequently using 3 volumes of elution BXT buffer (IBA). Elution fractions of 0.5 ml (EF#1 to EF#6) were collected and stored at -20 °C.
Protein samples were analyzed by polyacrylamide gel electrophoresis using precast gradient gels (NuPAGE, Bis-Tris, 4-12%, Thermo Fisher, Waltham, Massachusetts, USA).