TITLE: Purification of the PE2 nCas9-RT protein
AUTHORS: Donald   Rio

[DESCRIPTION]
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]
SECTION: A. Heat-shock Transformation
1. Thaw frozen competent cells on ice  until just thawed.

SECTION: A. Heat-shock Transformation
2. Gently mix the thawed competent cells (Rosetta 2 (pLysS)) by flicking the tube. 
SECTION: A. Heat-shock Transformation
3. Transfer 100 μl competent cells to a chilled culture tube.
SECTION: A. Heat-shock Transformation
4. Add 1 ng DNA plasmid to the cells.
SECTION: A. Heat-shock Transformation
5. Immediately return the tubes on ice  for 30 min 

SECTION: A. Heat-shock Transformation
6. Heat-shock the cells at 42 °C for 45 s.

SECTION: A. Heat-shock Transformation
7. Immediately place the tube on ice for 2 min.

SECTION: A. Heat-shock Transformation
8. Add 900 μl of cold SOC medium to the tube and incubate for 60 min at 37 °C with shaking   

SECTION: A. Heat-shock Transformation
9. Pellet the cells by centrifugation at 12000 x g, 2 min 

SECTION: A. Heat-shock Transformation
10. Remove the supernatant and plate onto agar plates containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol.

SECTION: A. Heat-shock Transformation
11. Incubate the plate at 37 °C for 60 min

SECTION: B. Protein Expression
12. Inoculate one colony into 50 ml LB containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol. Incubate 960 min on shaker   at 37 °C.

SECTION: B. Protein Expression
13. Transfer the overnight culture into 1 liter LB containing 50 μg/ml kanamycin and 34 μg/ml chloramphenicol to reach OD600 of 0.1.
SECTION: B. Protein Expression
14. Incubate at 37 °C with shaking   to reach OD600 of 0.6.

SECTION: B. Protein Expression
15. Add IPTG to a final concentration of 0.5 mM and grow for 960 min at 18 °C.

SECTION: B. Protein Expression
16. Harvest the cells by centrifugation at 5000 x g, 10 min, 4 °C 

SECTION: B. Protein Expression
17. Re-suspend the cell pellet with PBS, spin down and snap-freeze in liquid nitrogen for later purification.
SECTION: C. Protein Purification
18. Assemble a table column and fill the column with Ni-NTA resin to create a bed volume of 5 ml
SECTION: C. Protein Purification
19. Wash the column with 100 ml H2O.
SECTION: C. Protein Purification
20. Equilibrate the column with 5 CVs Ni-NTA loading buffer.
SECTION: C. Protein Purification
21. Thaw the cell pellet on ice until just thawed.

SECTION: C. Protein Purification
22. Re-suspend cell pellet (from 1 liter) with 35 mL lysis buffer
SECTION: C. Protein Purification
23. Sonicate for 5 min  (20 seconds on/off) and clarify by centrifugation at 25000 x g, 30 min 

SECTION: C. Protein Purification
24. Filter the supernatant through a 0.22 μm syringe filter.
SECTION: C. Protein Purification
26. Wash the resin with 100 ml Ni-NTA loading buffer followed by 50 ml Ni-NTA wash buffer.
SECTION: C. Protein Purification
27. Elute the protein in batch six times with 5 ml Ni-NTA elution buffer.
SECTION: C. Protein Purification
28. Analyze fractions by 7.5% SDS-PAGE and coomassie staining.
SECTION: C. Protein Purification
29. Collect relevant elution fractions, dilute into a low-salt buffer and filter through a 0.22 μm syringe filter
SECTION: C. Protein Purification
30. Load onto a 1 ml HiTrap heparin HP column pre-equilibrated in low-salt buffer. 
SECTION: C. Protein Purification
31. Elute the protein with a linear gradient of 100 mM to 1M KCl over 40 CVs. 
SECTION: C. Protein Purification
32. Analyze fractions by 7.5% SDS-PAGE and coomassie staining.
SECTION: C. Protein Purification
33. 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).
SECTION: C. Protein Purification
34. Make 3 µl protein sample aliquot and snap-freeze in liquid nitrogen. 
SECTION: C. Protein Purification
35. Store protein at -80 °C. 
SECTION: C. Protein Purification
25. Pour the supernatant into the table column in a single, continuous motion.
SECTION: A. Heat-shock Transformation
8.1. SOC medium
 

SECTION: B. Protein Expression
12.1. LB
 

SECTION: C. Protein Purification
20.1. Ni-NTA loading buffer 
 

SECTION: C. Protein Purification
22.1. Lysis buffer
 

Protease Inhibitor Cocktail (in 70% DMSO; 1000x)
 

SECTION: C. Protein Purification
26.1. Ni-NTA wash buffer
 

SECTION: C. Protein Purification
27.1. Ni-NTA elution buffer

 

SECTION: C. Protein Purification
29.1. Low salt buffer