Protocol

Abbreviation

uva_purification_refolding

Name

Protocol for purification in denaturing conditions

Laboratory name

University of Virginia

Type

purification

Description

1.Resuspend insoluble pellet in BB8MU ~30 min, RT, vortex
2. You can sonicate the pellet if it is still viscous
3.Spin down the lysate 35 000 rpm 4C 30 min – 1h collect samples of
soluble and
unsoluble fractions
4.Prepare column: Ni-NTA (3-7 ml of resin slurry, equlibrated with one
column volume of BB8MU)
5.Bind protein to the resin:
● betch method – 2 h on the rocker, RT
● apply supernatant on NiNTA column
● collect the sample of FT
6.Wash Ni-NTA column with 200-500 ml of WB8MU (can be done overnight)
7.Elute the protein with small volumes (2ml each) of EB8MU while checking
for protein by
Bradford Assay (10 μl of eluate with 200 μl of Bradford
reagent) collect sample for
SDS-PAGE analysis

Follow refolding protocol


RESUSPENSION BUFFER:
500 mM NaCl
50 mM Hepes ph=7.5
5% glycerol
8 M Urea

BINDING BUFFER:
500 mM NaCl
50 mM Hepes ph=7.5
5% glycerol
5mM Imidazole
8 M Urea

WASHING BUFFER:
500 mM NaCl
50 mM Hepes ph=7.5
5% glycerol
30 mM Imidazole
8 M Urea

ELUTION BUFFER:
500 mM NaCl
50 mM Hepes ph=7.5
5% glycerol
250 mM Imidazole
8 M Urea

Refolding protocol:

I. Column refolding
1. Take soluble fraction in 8M Urea BB and apply it on the NiNTA column.
2. Let it bind for 1h, RT
3. In 4C run through the column:
a) 100 ml of WB with 4 M Urea (50 ml of WB 8MU + 50 ml of WB)
b) 100 ml of WB with 2 M Urea (25 ml of WB 8MU + 75 ml of WB)
c) 100 ml of WB with 1 M Urea (12.5 ml of WB 8MU + 87.5 ml of WB)
d) 100 ml of WB with 0.5 M Urea (6.25 ml of WB 8MU + 93.75 ml of WB)
e) 100 ml of WB with 0 M Urea
4. Elute protein with EB 0 M Urea
5. Add rTEV protease (for each 100 mg of protein add 1mg of TEV)
6. Dialyse in DB 4C overnight
7. Apply protein to second Ni-NTA column to remove HisTag and uncut protein
8. Equilibrate Superdex 200 column with CB
9. Concentrate protein to a volume of 2ml
10. Run gel filtration
11. Concentrate protein to a final protein concentration of 10-20 mg/ml

II. Dialysis refolding

1.Take eluated protein and dialyse it against DB containing:
a) 4 M Urea
b) 2 M Urea
c) 1 M Urea
d) 0.5 M Urea
e) 0 M Urea
2. Add rTEV protease (for each 100 mg of protein add 1mg of TEV)
3. Dialyse in DB 4C overnight
4. Apply protein to second Ni-NTA column to remove HisTag and uncut protein
5. Equilibrate Superdex 200 column with CB
6. Concentrate protein to a volume of 2ml
7. Run gel filtration
8. Concentrate protein to a final protein concentration of 10-20 mg/ml

III. Fast dilution
1.Take eluated protein, and add the same volume of RB 0 M Urea --> 4 M
Urea, let it sit 2h,
then add:
a) 2 volumes of RB 0 M Urea --> 2 M Urea
b) 4 volumes of RB 0 M Urea --> 1 M Urea
c) 8 volumes of RB 0 M Urea --> 0.5 M Urea
2. Add rTEV protease (for each 100 mg of protein add 1mg of TEV)
3. Dialyse in DB 4C overnight
4. Apply protein to second Ni-NTA column to remove HisTag and uncut protein
5. Equilibrate Superdex 200 column with CB
6. Concentrate protein to a volume of 2ml
7. Run gel filtration
8. Concentrate protein to a final protein concentration of 10-20 mg/ml

If protein precipitates at any of those steps, spin it down, collect the
pellet, and apply pellet refolding protocol:

Pellet Refolding Protcol:

Prepare screen of different buffers for refolding or use commercial
protein refolding kit buffers

1. Take pellet and resuspend it in RB 8 M Urea
2. Divide into a lot of small volume samples (0.5-1 ml)
3. Dilute it rapidly into final concentration of 0.5 M Urea with buffer
screen
4. Leave in 4C ON
5. Check in the morning which conditions produce no precipitation
6. Leave it in RT for 2-3 h
7. Check in the which conditions produce no precipitation
8. Concentrate the protein
9. Run gel filtration using appropariate buffer as crystallization buffer
10. Concentrate protein to a final protein concentration of 10-20 mg/ml