TITLE: Human and mouse alpha-synuclein protein expression and purification
AUTHORS: andrew.west, arpine.sokratian

[DESCRIPTION]
The protocol for is designed for high-yield purification of recombinant α-synuclein monomer. It is recommended to always store the protein on ice, and once the purification process has started, it should not be stopped.

[STEPS]
SECTION: Transformation
1. Thaw down an aliquot of plasmid construct (pRK172) encoding WT-human-a-synuclein or mouse-a-synuclein 0.3 mg/mL on ice 

SECTION: Transformation
2. Thaw down on ice  an aliquot of BL21 (DE3) RIL competent E Coli cells
  

SECTION: Transformation
3. Add 1 µL  of plasmid construct to the thawed competent cells and gently mix by flicking the bottom of the tube with a finger a few times
 


SECTION: Transformation
3.1. Incubate the reaction mix on ice 

SECTION: Transformation
4. Perform heat-shock transformation 42 °C in water bath incubator with manually shaking at  
 



SECTION: Transformation
5. Immediately transfer the tube on ice and incubate for 1 min.
SECTION: Transformation
6. Add 1000 µL of SOC media to a chilled reaction
  

SECTION: Transformation
7. Incubate the bacteria   
 


SECTION: Transformation
8. Collect 50 ul of cell suspension (Tube #1 5% of cells) 
Centrifuge 950 cell suspension at 500 x g, 3 min, 10 °C 

Save the pellet with approximately 50 µL of media (Tube #2 95% of cells) 

8.1. Spread tubes #1 and #2 onto a selection plate and incubate overnight at 37 °C in bacterial incubator
 
 

SECTION: Transformation
7.1. Prepare sterile 10cm LB agar plate containing 0.1 mg/mL of ampicillin  
  

SECTION: Protein expression
9. Pick one colony and transfer into 10 mL LB media with 0.1 mg/mL of ampicillin
start in the morning (9:00 am)
  




SECTION: Protein expression
10. Transfer a starter culture to 2X2L flasks filled with 0.5L LB media with 0.1 mg/mL of ampicillin
5 mL to each flask 
 

SECTION: Protein expression
9.1. Incubate the bacteria   until it reaches OD 0.2-0.3
 

SECTION: Protein expression
11. Incubate the culture at the same conditions until it reaches OD 0.8 (use nanodrop or cuvette) (reaches optimal density at 6-7 pm)
SECTION: Protein expression
12. Induce protein expression by adding 0.05 millimolar (mM) IPTG, incubate at 18 °C for  720 minovernight
  

 


SECTION: Cell lysis
13. Collect the pellets by centrifugation (JA14 rotor) at 5000 x gx g at 4 °C for  10 min. Used 250 ml Beckman tubes
Usually get 10-12 g from 2L 
 

SECTION: Cell lysis
14. Add to pellets 80 ml of lysis buffer (total): 10 millimolar (mM) ThisHCl pH 7.6, 750 millimolar (mM) NaCl, 1 millimolar (mM) EDTA, 1 millimolar (mM) PMSF (add just before using, have aliq frozen 0.1 Molarity (M)), protease inhibitors (use MAXI version, need only one tablet); 
    
  
  

SECTION: Cell lysis
15. Carefully resuspend the pellets to homogenize the solution
SECTION: Cell lysis
16. While waiting on water to get to the boiling point sonicate the lysates (use thick prob-tip) for 1 min, 30%, 15 s ON 30 s OFF of amplitude then go to next falcon, had 3 falcons (repeat 3 times, avoid overheating)

SECTION: Cell lysis
15.1. Heat up 1 Lof water in a high temperature resistant glass beaker (turn heat to the max on the magnetic stirrer)

SECTION: Cell lysis
17. After sonication samples need to get boiled thereby put the falcon tubes into glass beaker and boil for 25 min. Use tweezers to pull out the tubes

SECTION: Cell lysis
18. Transfer boiled homogenates into new 50 mL falcon tubes; chill down suspensions at room temperature for 20 min
SECTION: Cell lysis
19. Prepare 4 L of buffer 10 millimolar (mM) TrisHCl pH 7.6, 50 millimolar (mM) NaCl, 1 millimolar (mM) EDTA, 1 millimolar (mM) PMSF for dialysis 

SECTION: Cell lysis
20. Centrifuge the homogenates at 20000 x gfor 60 min at 4 °C 

SECTION: Cell lysis
21. Filter the supernatant using 0.45 um filter unit   

SECTION: Protein purification (anion-exchange chromatography)
23. After a night of dialysis (4 °C slow mixing) collect the suspension into 100 mL glass bottle (filter the sample before running on the column, 0.22 um filter). 

SECTION: Cell lysis
22. Transfer filtered supernatant into dialysis bag which is: SnakeSkin Dialysis tubing, 3.5K MWCO, 35 mm dry I.D., 35 feet.
Measure the dialysis tube taking into consideration that 5 cm length of tube holds 48 mL of the sample (plus 2.5cm at each end for closure). Clip the tube using green clips, make sure it does not leak. 
Place the dialysis bag into4 Lplastic beaker filled with dialysis buffer, incubate overnight on magnetic plate on the slow mode (Chromatography fridge) 
  

 


SECTION: Protein purification (anion-exchange chromatography)
24. Column - HiPrep Q HP 16/10 column 1x20 ml (stored in 70% ethanol);  



SECTION: Protein purification (anion-exchange chromatography)
25. Load 80 mL of suspension and then washed with 100 ml 50 millimolar (mM) 
NaCl 10 millimolar (mM) TrisHCL, 300 mLof gradient elution (0-100%), 2 ml/min flow rate. Collected samples using fraction collector 2, every fraction 4 ml (use 10 ml glass tubes)

SECTION: Protein purification (anion-exchange chromatography)
29. Analyze the fractions eluted at 250-350 mM salt (20 RFU conductivity) though SDS-PAGE (stain with Coomassie). 
Combine 10 µL of each fraction with 10 µL of 2X laemmli buffer and analyze fractions by SDS-PAGE with 4–20% gradient gels, followed by coomassie staining/destaining

SECTION: Protein purification (anion-exchange chromatography)
24.1. Wash the column 2V of miliQ degassed water 
SECTION: Protein purification (anion-exchange chromatography)
24.2. Wash the column with 2V of STARTING BUFFER  10 millimolar (mM) TrisHCl pH 7.6, 50 millimolar (mM) NaCl
SECTION: Protein purification (anion-exchange chromatography)
24.3. Activate with 1V of 10 millimolar (mM) TrisHCl pH 7.6, 1 Molarity (M) NaCl 
SECTION: Protein purification (anion-exchange chromatography)
24.4. Equilibrate with 3V of starting buffer 
SECTION: Protein purification (anion-exchange chromatography)
26. Place supernatant into channel A1 (was previously use for starting buffer, do not generate bubbles)
SECTION: Protein purification (anion-exchange chromatography)
27. Place starting buffer in channel A2 (clean the tubing using the program mode)
SECTION: Protein purification (anion-exchange chromatography)
28. Place elution buffer in channel B1 (10 millimolar (mM) TrisHCl pH 7.6, 1 Molarity (M) NaCl)
Collected samples using fraction collector 2, every fraction 4 ml (use 10 ml glass tubes); 
SECTION: Protein purification (anion-exchange chromatography)
30. Measure A280/260 for the fractions containing single a-syn band, avoid collecting samples with A280/260 > 0.85 
SECTION: Protein purification (anion-exchange chromatography)
31. Combine the evaluated factions and measure total protein concentration using nanodrop. 
SECTION: Protein purification (anion-exchange chromatography)
32. Dialyze with 4 Lof   10 millimolar (mM) TrisHCl pH 7.6, 50 millimolar (mM) NaCl (follow instruction for dialysis)

SECTION: Protein concentration
34. Concentrate dialyzed protein sample to approximately 30 mg/mL aliquot
Add 3 µL of 10x diluted aliquot in PBS onto nanodrop pedestal; 
Parameters: 
 - other proteins; coefficient extinction: 5.98; MW: 14.4 kDA (for wild-type human a-synuclein)
 - other proteins; coefficient extinction: 7.45; MW: 14.4 kDA (for wild-type mouse a-synuclein)
Perform two measurements and confirm <10% standard error between two measurements
If necessary, prepare 20X and 30X dilutions to confirm findings. 
 




Prepare the ultra-concentration system
SECTION: Further purification
33. Repeat section 'Protein purification (anion-exchange chromatography)' for the further fractionation of the purified preparation  

SECTION: Protein concentration
35. Use 50 mL ultra centrifugation units with 3K cutoff  

SECTION: Protein concentration
38. Centrifuge at 5000 x g at 4 °C for 5 min , JA10 rotor

SECTION: Protein concentration
36. Wash off the unit with miliQ water through centrifugation at 5000 x g at 4 °C for 5 min , JA10 rotor

SECTION: Protein concentration
37. Load first 15 mL of the sample into ultracentrifugation unit (max load of the unit is approx. 15 mL) 

SECTION: Protein concentration
40. Store at -80 °C. Yield should be approximately 80 mg per 2 L culture

SECTION: Protein concentration
39. Resuspend concentrated sample, add more of protein sample and concentrate until the total volume is ~5 mL

SECTION: Endotoxin removal
42. Collect the eluate into 5 mL endotoxin-free tube and save 2 aliquots (10 µLand 50 µL) for protein concentration and endotoxin measurements
  

SECTION: Endotoxin removal
41. Follow instructions for   with modifications
For a more successful endotoxin removal, add 1 mL of 
  before the regeneration process

SECTION: Endotoxin quantification
43. Follow instructions for Endotoxin detection kit LAL Genscript