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.