A streamlined tandem affinity purification of His-MBP-SpyCas9, without buffer exchange, suitable for in vitro cleavage applications

We present a streamlined protocol for the purification of recombinant doubly-tagged His-MBP-SpyCas9 protein utilizing a dual affinity chromatography approach whereby the elution volume from immobilized metal ion affinity chromatography (IMAC) is loaded directly onto maltose-binding protein (MBP) affinity chromatography. This protocol, by optimizing the buffer composition throughout the process, eliminates the need for buffer exchanges thereby reducing the risk of protein precipitation. The purification process, from bacterial harvest to final product, can be completed within a single working day. The purified Cas9 protein is suitable for in vitro cleavage assays as validated using sgRNA/complementary dsDNA-reporter oligonucleotides. In fact, the elution buffer from the MBP binding column is suitable for storage and the purified protein can be stored/aliquoted at -20 °C after the addition of glycerol, to be used directly in vitro cleavage assays without additional processing.