TITLE: LRRK2 RCKW Protein Purification
AUTHORS: David Snead, Yu Xuan Lin, Mariusz  Matyszewski

[DESCRIPTION]
Protein purification protocol for tag-less LRRK2RCKW as done by Leschziner and Reck-Peterson Labs. Same protocol can be used to purify LRRK1RCKW as well.

Original Protocol by David Snead. Modified by Yu Xuan Lin and Mariusz Matyszewski for publication.

[STEPS]
SECTION: Day 1: Protein Pellet Lysis
1. Ready the Ultracentrifuge and cool it down to  4 °C 

SECTION: Day 1: Protein Pellet Lysis
2. Make Lysis Buffer (check Materials section)
SECTION: Day 1: Protein Pellet Lysis
3. Thaw protein pellets on ice 

SECTION: Day 1: Protein Pellet Lysis
4. Resuspend each pellet in 40 mL

 

SECTION: Day 1: Protein Pellet Lysis
5. Homogenize each pellet with Dounce homogenizer on ice 

SECTION: Day 1: Protein Pellet Lysis
5.1. 15 plunges loose, followed by 17 plunges tight.

 

SECTION: Day 1: Protein Pellet Lysis
6. Pour lysate into rotor tubes (pre-chilled, clean, check for cracks; we used Ti70 tubes)
SECTION: Day 1: Protein Pellet Lysis
6.1. Make sure they are balanced. Use Lysis buffer to balance if needed.
SECTION: Day 1: Protein Pellet Lysis
7. Spin lysate in ultracentrifuge 50000 rpm, 88 min, 4 °C 

SECTION: Day 1: Protein Pellet Lysis
8. Prep the rest of Day 1 buffers while waiting
SECTION: Day 1: Ni-NTA gravity column
9. Start equilibrating 12 mL of Ni-NTA beads with Lysis buffer when there is about 30 mins left on the centrifuge

SECTION: Day 1: Ni-NTA gravity column
9.1. We equilibrated by resuspending 6 mL  in a 15 mL falcon tube, twice. 
They were spun down at 1000 rpm, 2 min, 4 °C , each time getting rid of liquid and doing a 50:50 resuspension.

SECTION: Day 1: Ni-NTA gravity column
10. After ultracentrifuge is done, pour the supernate into 3x 50 mL falcon tubes.
SECTION: Day 1: Ni-NTA gravity column
11. Add 4 mL to 40 mL of supernate. Bring up to 50 mL 

SECTION: Day 1: Ni-NTA gravity column
12. Incubate in cold room, while rotating "hot-dog style".   

SECTION: Day 1: Ni-NTA gravity column
13. After incubation, add onto a gravity column. Wash with remaining Lysis Buffer, followed by 200 mL 

SECTION: Day 1: Ni-NTA gravity column
14. Elute with 50 mL 
Make sure to resuspend the beads with stopper closed on the column. Might need multiple repeats of resuspension for best results. Leave column dry afterwards.
SECTION: Day 1: Ni-NTA gravity column
15. Dilute the solution to 250 millimolar (mM) by adding 50 mL 

SECTION: Day 1: Ni-NTA gravity column
16. Syringe filter the solution.
SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
17. Use a 5 mL HiTrap SP FF column. Pump wash FPLC with Buffer A and Buffer B. Equilibrate the column with Buffer A.
SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
18. Apply sample onto the column with a sample pump
SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
19. Run a gradient program, 250 millimolar (mM) to 2.5 Molarity (M) , 0% B to 100% B.
SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
20. Combine protein fractions into a 50 mL Falcon Tube. Check protein concentration; should be around 1.1 micromolar (µM) 

SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
20.1. Afterwards dilute to 600 millimolar (mM) 

SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
20.2. Bring up to 50 mL 

SECTION: Day 1: FPLC Cation Exchange and Overnight TEV cleavage
21. Add TEV, and incubate overnight at 4 °C while rotating "hot-dog style".

 


SECTION: Day 2: FPLC HisTrap Column
22. Make Day 2 buffers. Make sure to degas the Storage Buffer.
SECTION: Day 2: FPLC HisTrap Column
23. Put the 5 mL HisTrap column on the FPLC. Pump wash the FPLC with Buffer 0 and Buffer 300. Equilibrate the column with Buffer 0.
SECTION: Day 2: FPLC HisTrap Column
24. Run a Step Gradient Program
 


SECTION: Day 2: FPLC HisTrap Column
25. Combine protein elution into a 15 mL tube before concentrating for Size Exclusion Column
SECTION: Day 2: SEC Column
26. Equilibrate a S200I 10/300 column with the Storage Buffer.
SECTION: Day 2: SEC Column
27. Concentrate the protein to 1 mL using 100 kD cutoff concentrators. Should be done nearing the end of the column equilibration.

 

SECTION: Day 2: SEC Column
27.1. After concentrating, filter the protein using a  0.1 uM spin filter. Spin at max speed for 2 min at 4 °C 

SECTION: Day 2: SEC Column
28. Inject filtered sample and run elution program.
SECTION: Day 2: SEC Column
29. Collect and concentrate the protein to about 500 µL . Concentration should be around 25-30 micromolar (µM) after concentrating.

 

SECTION: Day 2: SEC Column
30. Aliquot and freeze protein. Make sure to check pureness of protein as well.