Jul 26, 2022
Purification of the PE2 nCas9-RT protein
- 1University of California, Berkeley
External link: https://doi.org/10.7554/eLife.79208
Protocol Citation: Donald Rio 2022. Purification of the PE2 nCas9-RT protein. protocols.io https://dx.doi.org/10.17504/protocols.io.b4yxqxxn
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 12, 2022
Last Modified: June 01, 2024
Protocol Integer ID: 58103
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
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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
Materials
| Item | Vendor | Catalog # | |
| Tryptone | U.S. Biotech Sources, LLC | T01PD-500 | |
| Yeast extract | BD Bacto | 288620 212750 | |
| NaCl | Fisher Scientific | S271 | |
| KCl | Macron Fine Chemicals | 6858-06 | |
| MgCl2 | Fisher Scientific | BP214 | |
| MgSO4 | Fisher Scientific | M63 | |
| Glucose | Sigma | G8270 | |
| Kanamycin | Goldbio | K-120-SL25 | |
| Chloramphenicol | Goldbio | C-105-5 | |
| IPTG | Goldbio | I2481C | |
| HEPES | Omnipur | 5320 | |
| Imidazole | Sigma | 12399 | |
| DTT | Goldbio | DTT100 | |
| PMSF | Sigma | P7626 | |
| Ni-NTA Superflow | QIAGEN | 30410 | |
| HiTrap heparin HP | GE Healthcare | 17040601 | |
| Spin-X UF 20 50 kDa MWCO | Corning | 431488 | |
| DMSO | Fisher Scientific | BP231-100 | |
| Leupeptin | Millipore | 634987 | |
| Pepstatin | Sigma | P5318 | |
| Chymostatin | Sigma | C7268 | |
| Aprotinin | Sigma | A6279 | |
| Antipain | Millipore | 6C0417 |
A. Heat-shock Transformation
A. Heat-shock Transformation
2h 34m 45s
2h 34m 45s
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 00:30:00
30m
Heat-shock the cells at 42 °C for 00:00:45 .
45s
Immediately place the tube On ice for 00:02:00 .
2m
Add 900 μl of cold SOC medium to the tube and incubate for 01:00:00 at 37 °C with shaking 175 rpm
1h
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 12000 x g, 00:02:00
2m
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 Overnight
1h
B. Protein Expression
B. Protein Expression
16h 10m
16h 10m
Inoculate one colony into 50 ml LB containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Incubate Overnight on shaker 175 rpm at 37 °C .
16h
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 175 rpm to reach OD600 of 0.6.
Add IPTG to a final concentration of 0.5 mM and grow for 16:00:00 at 18 °C .
16h
Harvest the cells by centrifugation at 5000 x g, 4°C, 00:10:00
10m
Re-suspend the cell pellet with PBS, spin down and snap-freeze in liquid nitrogen for later purification.
C. Protein Purification
C. Protein Purification
35m
35m
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 On 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 00:05:00 (20 seconds on/off) and clarify by centrifugation at 25000 x g, 00:30:00
35m
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.