Glutathione sepharose 4b gs4b beads

Point mutations were introduced using Fast Site-Directed Mutagenesis Kit in the Streptococcus pyogenes Cas9 gene and verified by DNA sequencing. The cDNA of full-length xCas9 was sub-cloned into the bacterial expression vector pGEX-6P-1 (GE Healthcare, with an N-terminal GST tag). WT and mutants of SpCas9 proteins were expressed in E.coli C43 (DE3) cells. Expression of the recombinant proteins was induced by 0.3 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16 °C. After overnight induction, the cells were collected by centrifugation, xCas9 was resuspended in buffer A (25 mM Tris-HCl, pH 8.0, 1 M NaCl, 3 mM DTT) supplemented with 1 mM protease-inhibitor PMSF (phenylmethanesulphonylfluoride, Sigma). The cells were subjected to lysis by sonication and cell debris was removed by centrifugation at 23,708 × g for 40 min at 4 °C. The lysate was first purified using glutathione sepharose 4B (GS4B) beads (GE Healthcare). The beads were washed and the bound proteins were cleaved by precision protease in buffer B (25 mM Tris-HCl, pH 8.0, 300 mM NaCl, 3 mM DTT) overnight at 4 °C to remove the GST tag. The cleaved xCas9 protein was eluted from GS4B resin and further fractionated by heparin sepharose column and ion exchange chromatography via FPLC (AKTA Pure, GE Healthcare).