Purification of the PE2 nCas9-RT protein
Donald Rio
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Abstract
This protocol describes the process of expressing and purifying the nicking Cas9-MMLV RT fusion protein for prime editing.
Protocol overview
A. Heat-shock Transformation
B. Protein Expression
C. Protein Purification
Steps
A. Heat-shock Transformation
Thaw frozen competent cells On ice until just thawed.
Gently mix the thawed competent cells (Rosetta 2 (pLysS)) by flicking the tube.
Transfer 100 μl competent cells to a chilled culture tube.
Add 1 ng DNA plasmid to the cells.
Immediately return the tubes On ice for 0h 30m 0s
Heat-shock the cells at 42°C for 0h 0m 45s.
Immediately place the tube 42On ice for 0h 2m 0s.
Add 900 μl of cold SOC medium to the tube and incubate for 1h 0m 0s at 37°C with shaking 175rpm
SOC medium
| A | B |
|---|---|
| Tryptone | 2 % |
| Yeast extract | 0.5 % |
| NaCl | 10 mM |
| KCl | 2.5 mM |
| MgCl2 | 10 mM |
| MgSO4 | 10 mM |
| Glucose | 20 mM |
Pellet the cells by centrifugation at 12000x g
Remove the supernatant and plate onto agar plates containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol.
Incubate the plate at 37°C for 1h 0m 0s
B. Protein Expression
Inoculate one colony into 50 ml LB containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Incubate 16h 0m 0s on shaker 175rpm at 37°C.
LB
| A | B |
|---|---|
| Tryptone | 2 % |
| Yeast extract | 0.5 % |
| NaCl | 10 mM |
Transfer the overnight culture into 1 liter LB containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol to reach OD600 of 0.1.
Incubate at 37°C with shaking 175rpm to reach OD600 of 0.6.
Add IPTG to a final concentration of 0.5 mM and grow for 16h 0m 0s at 18°C.
Harvest the cells by centrifugation at 5000x g,4°C
Re-suspend the cell pellet with PBS, spin down and snap-freeze in liquid nitrogen for later purification.
C. Protein Purification
Assemble a table column and fill the column with Ni-NTA resin to create a bed volume of 5 ml
Wash the column with 100 ml H2O.
Equilibrate the column with 5 CVs Ni-NTA loading buffer.
Ni-NTA loading buffer
| A | B |
|---|---|
| HEPES-KOH pH 7.6 | 25 mM |
| KCl | 150 mM |
| Imidazole | 20 mM |
| DTT | 1 mM |
| PMSF | 1 mM |
Thaw the cell pellet 18On ice until just thawed.
Re-suspend cell pellet (from 1 liter) with 35 mL lysis buffer
Lysis buffer
| A | B |
|---|---|
| HEPES-KOH pH 7.6 | 25 mM |
| KCl | 1 M |
| Imidazole | 20 mM |
| DTT | 1 mM |
| PMSF | 1 mM |
| Protease Inhibitor Cocktail | ×1 |
Protease Inhibitor Cocktail (in 70% DMSO; 1000x)
| A | B |
|---|---|
| Leupeptin | 0.5 mg/ml |
| Pepstatin | 0.5 mg/ml |
| Chymostatin | 0.5 mg/ml |
| Aprotinin | 0.5 mg/ml |
| Antipain | 0.5 mg/ml |
Sonicate for 0h 5m 0s (20 seconds on/off) and clarify by centrifugation at 25000x g
Filter the supernatant through a 0.22 μm syringe filter.
Pour the supernatant into the table column in a single, continuous motion.
Wash the resin with 100 ml Ni-NTA loading buffer followed by 50 ml Ni-NTA wash buffer.
Ni-NTA wash buffer
| A | B |
|---|---|
| HEPES-KOH pH 7.6 | 25 mM |
| KCl | 150 mM |
| Imidazole | 40 mM |
| DTT | 1 mM |
| PMSF | 1 mM |
Elute the protein in batch six times with 5 ml Ni-NTA elution buffer.
Ni-NTA elution buffer
| A | B |
|---|---|
| HEPES-KOH pH 7.6 | 25 mM |
| KCl | 150 mM |
| Imidazole | 500 mM |
| DTT | 1 mM |
| PMSF | 1 mM |
Analyze fractions by 7.5% SDS-PAGE and coomassie staining.
Collect relevant elution fractions, dilute into a low-salt buffer and filter through a 0.22 μm syringe filter
Low salt buffer
| A | B |
|---|---|
| HEPES-KOH pH 7.6 | 25 mM |
| KCl | 100 mM |
| DTT | 1 mM |
| PMSF | 1 mM |
Load onto a 1 ml HiTrap heparin HP column pre-equilibrated in low-salt buffer.
Elute the protein with a linear gradient of 100 mM to 1M KCl over 40 CVs.
Analyze fractions by 7.5% SDS-PAGE and coomassie staining.
Pool peak elution fractions and concentrate using a Spin-X UF 20 50 kDa MWCO to 8 mg/ml (determine protein concentration by UV at wavelength of 280 nm).
Make 3 µl protein sample aliquot and snap-freeze in liquid nitrogen.
Store protein at -80 °C.
