The homologous Cas9 was fused downstream of the Maltose binding protein and a TEV protease cleave site. For bacterial expression of the recombinant proteins Cas9, corresponding plasmid was transformed into BL21-Plys (DE3)- competent cells (Stratagene). Expression of the recombinant proteins was induced by subculturing 5 ml starter culture into 1000 ml LB, adding at OD600 》 0.5－0.7 with 0.5 mM IPTG (isopropyl-1-thio-D-galactopyranoside) and shaking at 18oC for 20 hours. For protein purification, bacterial cells were harvested from 6L induced culture and resuspended in 100mL column buffer (20mM Tris HCl pH7.5, 5% Glycerol , 0.5M NaCl) containing 2 mM PMSF and protease inhibitor cocktails. Then the bacterial cells were lysed by sonication and supernatant from the lysed cultures were collected for subsequent column purified using Ni-resin.
Competent cell: BL21Plys(DE3) Grow media: LB
Antibiotic: 50ug/ml Kan and 34ug/ml chloramphenicol
OD (600) at induction: > 0.5-0.7 Grow temperature: 37oC
Induction Temperature: 18oC (20 hours)
TGI buffer: 20mM Tris-HCl, pH 7.5, 5% Glycerol, 0.5M NaCl, 1L
Lysis buffer: 20mM Tris-HCl, pH 7.5, 5% Glycerol, 0.5MNaCl, 2mM PMSF, protease inhibitors, 100ml
Elution buffer-I: 20mM Tris-HCl, pH 7.5, 5% Glycerol, 0.5M NaCl. 250mM Imidazole, 100ml
Wash buffer: 20mM Tris-HCl, pH7.5, 5% Glycerol, 0.5M NaCl, 25mM Imidazole, 500ml
Ni-Column charging buffer: 100mM NiCl
Dialysis buffer: 20mM HEPE, pH 7.5, 150mM KCl
Gel filtration buffer: 20mM HEPE, pH 7.5, 150mM KCl
SP Sepharose buffer A: 20mM HEPE, pH 7.5, 5% glycerol, 150mM KCl;
SP Sepharose buffer B: 1M KCl;
1. Add 100ml lysis buffer to each 6L harvest cells, sonicate
2. Spin down 30min at 18K;
3. Pre-equilibrate Ni resin in TGI buffer
4. Add 10ml of the pre-equilibrated Ni resin to each supernatant resulted from 3L LB culture;
5. Incubate the mixed Ni resin at 4oC for at least 2 hours.
6. Wash the Ni resin with 10CV wash buffer
7. Load the Cas9-MBP protein mix to an empty Column.
8. Wash the column with 10CV wash buffer
9. Elute the protein with 3CV elution buffer
10. Collect loading through (1.5ml/tube)
11. Run SDS gel;
12. Dialysis SyCas9 in the dialysis buffer at 4oC over night with TEV protease (0.5ml).
13. Load the TEV cleaved SpCas9 to pre-washed 5ml SP Sepharose
14. 10CV wash and gradient elution
13. Concentrate SyCas9 (to >5mg/ml)
14. Load to Superdex200 10/300 increase (new column) gel filtration column.
15. Run SDS gel for the fractions
16. Pool fraction 10 to 12 and concentrated to 2.98mg/ml (23uM)