(a) LwaCas13a protein expression and purification
workflow. The TwinStrep-SUMO-LwaCas13a protein expression plasmid is
first transformed into competent Rosetta E. Coli cells. After
antibiotic selection and initial growth, expression is induced by IPTG.
Following growth, cells are harvested and lysed. The recombinant protein is
subsequently enriched from the total cell protein by affinity Streptactin
purification. The TwinStrep-SUMO tag is then cleaved by SUMO protease to obtain
native LwaCas13 protein. The enzyme is further purified by ion-exchange and size
exclusion chromatography on an FPLC system.
(b) FPLC Chromatograms. The graph shows a representative
chromatogram of ion-exchange (IEC, top image) and size-exclusion chromatogram
(SEC, bottom image) for LwaCas13a. The UV-absorbance in mAU is plotted against
the elution volume in mL. The NaCl gradient for ion-exchange chromatography is
shown by the green line. The red boxes indicate the protein-containing fractions
that were pooled and concentrated.
(c) Coomassie stained SDS-PAGE gel of protein fraction The
progress of protein purification is shown on a Coomassie stained SDS-PAGE gel.
The fractions are L: Ladder, 1: Cell lysate, 2: Cleared cell lysate, 3: Cell
pellet after clearing of lysate, 4: Flow-through following StrepTactin
batch-binding, 5: StrepTactin resin before SUMO protease cleavage, 6: Eluted
fraction post SUMO protease cleavage, 7: Concentrated sample post ion-exchange
chromatography, 8: Final product after size-exclusion chromatography.