3.1 Production and purification of rSaCas9
To provide sufficient amounts of recombinant SaCas9 for subsequent activity assays, the target protein was produced by coupled in vitro transcription/translation in the cell-free BYL lysate. The ready-to-use BYL reaction contains all required compounds for in vitro transcription and translation, including the T7 polymerase initiating transcription [12]. Only the template vector carrying the expression cassette encoding rSaCas9 has to be added, allowing easy and rapid protein biosynthesis. The recombinant SaCas9 enzyme with additional NLS and Strep-tag II (Figure S2) has a calculated molecular mass of 129.2 kDa. SDS–PAGE analysis of crude BYL producing either rSaCas9 or eYFP as a positive control demonstrated the successful production of both recombinant proteins with apparent molecular masses of 130 kDa and 32 kDa, respectively (Figure 2). Both proteins are clearly visible as prominent bands within the background of tobacco host proteins. The whole process lasted 48 h, but by 24 h, the production of both recombinant proteins could be observed.
The rSaCas9 nuclease was purified using the C-terminally attached Strep-tag II for affinity chromatography on a Strep-Tactin matrix. This single-step purification protocol yielded a highly pure rSaCas9 apoenzyme, as no major contaminating bands were visible in the elution fractions (EF#1-EF#6) on the acrylamide gel. The highest enzyme concentrations were observed in elution fractions EF#2 and EF#3 (Figure 3). The purified rSaCas9 protein has an apparent molecular mass of 130 kDa, demonstrating the integrity of the enzyme and the absence of proteolytic degradation. The presence of an rSaCas9 moiety in the pellet fraction suggested that a proportion of rSaCas9 is insoluble and thus precipitates. The flow-through and wash fractions also contained rSaCas9, indicating that a portion of SaCas9 was either not bound or only weakly bound to the matrix. Purified protein from EF#2 was used for subsequent activity assays on target sequences.