Protocol

Abbreviation

UTSW_standard purification

Name

UTSW_standard purification

Laboratory name

University of Texas Southwestern Medical Center

Type

purification

Description

Purification

Cell disruption:
1. Spin 1L bacterial cultures at 4000rpm 40mins at 4 ºC.
2. Prepare BB buffer.
3. Decant the media (incubate with bleach for at least 1hour before you pour it into sink)
4. Resuspend the cell plate in cold BB buffer (35ml of BB per 1L of bact. Culture), transfer the suspension into 50mL plastic tubes, label and freeze at -80 ºC.
5. Thaw cells in cold water and protease inhibitors (1mM final concentration of PMSF and benzamidine)
6. Sonicate on ice for 5X30s with a 30s rest in between. Repeat if the lysate is still viscous.
7. Take 30 µL sample for SDS-PAGE, spin 20mins (microfuge) at 13000rpm, separate soluble fraction from sediment, label SUP and SED and add 15 µL of loading buffer with DTT and heat at 99 ºC for 3mins.
8. Freeze the lysate at -80 ºC.

Buffer Preparation:

Prepare buffers for purification, check pH and filter them.

BB buffer:
500mM NaCl (50ml/500ml of 5M NaCl)
5% glycerol (25ml/500ml of 100% glycerol)
50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5)
5mM imidazole (2.5ml/500ml of 1M imidazole)

WB buffer:
500mM NaCl (50ml/500ml of 5M NaCl)
5% glycerol (25ml/500ml of 100% glycerol)
50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5)
30mM imidazole (15ml/500ml of 1M imidazole)

EB buffer:
500mM NaCl (50ml/500ml of 5M NaCl)
5% glycerol (25ml/500ml of 100% glycerol)
50mM HEPES pH 7.5 (25ml/500ml of 1M HEPES pH 7.5)
250mM imidazole (175ml/500ml of 1M imidazole)

5mM β-mercaptoethanol must be added to all buffers before use (117 µL/500m of 100% β-mercaptoethanol)

Dialysis Buffer # 1
500mM NaCl (400ml/4L 5M NaCl)
5% glycerol (200ml/4L 100% glycerol)
10mM HEPES pH 7.5 (80ml/4L 0.5M HEPES pH 7.5)

Same buffer for Dialysis Buffer # 2(1L) and Gel filtration buffer(2L)
200mM NaCl (120ml/3L 5M NaCl)
100mM HEPES pH 7.5 (600ml/3L 0.5M HEPES pH 7.5)
5% glycerol (150ml/3L 100% glycerol)


First affinity chromatography:
1. Spin the lysate at 12000 rpm at 4 ºC for 40min and filter it through 0.8µm filters.
2. Prepare columns as follows:
- DE52 (Whatman): mix 10gms of DE52 in 100ml of 2.5M NaCl, add to large column and equilibrate with 40ml of BB buffer
- Ni resin (Quiagen): add 5ml bed volume to small column and equilibrate with 20ml BB buffer.
- Place column in series (DE52 → Ni resin)
3. Save the sediment after centrifugation at -80ºC (can be removed after the purification is over and OK)
4. Apply the clarified lysate to upper DE52 column and let it flow through both columns, collect flow-through fraction (FT)
5. Take 30 µl sample for SDS-PAGE, label FT and add 15 µl of loading buffer with DTT and heat at 99ºC for 3min.
6. Add 2x10 ml of BB buffer to DE52 column ( don’t collect the flow-through fraction)
7. Remove the DE52 column and work further only with Ni column.
8. Wash Ni column with 40ml WB buffer and collect WB fraction.
9. Take 30µl sample for SDS-PAGE, label WB and add 15µl of loading buffer with DTT.
10. On the ‘last drop’ of WB perform Bradford assay to determine if all protein were washed out. If not wash with WB buffer further until OD595nm is ~ 0.05. Don’t collect fraction.
11. Let the column dry briefly, add 2ml of EB buffer and let sit for 1min. Elute the protein sequentially with 10ml of EB buffer while checking protein content by Bradford assay. Stop elution when OD595nm is ~ 0.05. Collect all elution into one 50ml tube, label EB (ideally the volume should not exceed 50ml)
12. Take 30µl sample for SDS PAGE, label EB and add 15µl of loading buffer with DTT and heat at 99ºC for 3min.
13. Add EDTA to EB fraction to final concentration 1mM.
14. Add TEV protease to EB fraction(follow the instructions on box with TEV prep)
15. Dialyze O/N into 4L of Dialysis buffer #1 at RT. Do not add more than 80ml of sample into 4L dialysis buffer.

Second affinity chromatography:

1. Take 30µl sample for SDS PAGE from dialysis bag, label TEV and add 15µl of loading buffer with DTT and heat at 99ºC for 3min.
2. Run SDS-PAGE gel with samples from purification and after TEV cleavage.
3. If gel shows that His-Tag is cleaved out you can proceed to second affinity chromatography step. If cleavage was not sufficient, allow the protease digest further.
4. Add protease inhibitors to the content of dialysis tube (1mM final concentration of PMSF and benzamidine).
5. Add 6ml of Ni resin pre-equilibrated in BB buffer and shake at 4ºC for 40mins.
6. Pour the suspension into small column and collect the flow through (label FT2)
7. Then wash the column by 5ml of BB several times until all protein is washed out (as determined by Bradford assay), collect as BB2.
8. Take 30µl sample for SDS PAGE from FT2 and BB2, add 15µl of loading buffer with DTT and heat at 99 ºC for 3mins.
9. Dialyze FT2+BB2 O/N into 4L of dialysis buffer #2 at 4ºC. Do not add more than 80ml of sample into 4L of dialysis buffer.
Gel filtration:
Setup HPLC column – Superdex 75 HR 10/30.
Collect elutes and run SDS-PEGE and

Protein concentration:

1. Run SDS PAGE gel with samples from purification and after TEV cleavage.
2. While running gel, start protein concentration by ultra filtration using Vivaspin concentrators. Concentrate protein to ~50mg/ml or until precipitation occurs, remove from concentrator and determine volume. Wash concentrator with equal volume of 10mM HEPES pH 7.5 and pool the 2 fractions.
3. Spin at 13000rpm and determine concentration by OD280nm.
4. Take 10µl sample for SDS PAGE add 5µl of loading buffer with DTT and heat at 99 ºC for 3mins.
5. Take 10µl sample for SDS PAGE add 5µl of loading buffer without DTT and heat at 99 ºC for 3mins.
6. Take 10µl sample for native PAGE add 5µl of native loading buffer.
7. Aliquot the protein, label and store at -8 ºC

Protein analysis before crystallization:

Run SDS PAGE gel and native electrophoresis gel.