Protein expression and purification

IL-10 purification protocols

Hi5 Secreted Protein Precipitation Method

For Monomeric IL-10 and receptors

Cell Growth

Hi5 (insect cells are grown in InsectXpress media (Lonza Bioscience) in 2 litre plastic wide- bottomed Fernbach culture flasks:

(https://www.thermofisher.com/order/catalog/product/4105-2800?SID=srch-srp-4105-2800#/4105-2800?SID=srch-srp-4105- 2800).

They were grown under constant rotation (125 rpm) on a shaking table in a warm room (27- 28 ºC). 1 litre of cells was infected with 1ml of P1 virus at a cell density of 2 x 10⁶ per ml. The cells were allowed to grow for 48 hours prior to harvesting.

Purification:

Materials:

• ROOM TEMPERATURE large volume centrifuge (e.g. Beckman Avanti J-20 & JLA-8.100 rotor)
• 1M Nickel Chloride
• 5M Calcium Chloride
• 1M Tris pH 8.0
• Millipore type HA filters
• Ni-NTA slurry
• Room temperature magnetic stir plate
• 1M imidazole pH 7.2 in 1X HBS
  • 10X HBS Recipe (→ Dilute to 1X for working concentration)
    • 100 mM HEPES
    • 1.5 M Sodium Chloride
    • 0.2% Sodium Azide
    • Adjust pH to 7.2
• 1L centrifuge bottles

All steps should be carried out at room temperature. If the Tris gets cold, the pH will change.

1. Spin cells in 1L bottles, 2000 rpm for 15 min. AT ROOM TEMPERATURE in centrifuge

2. Pour cell supernatant into a large (with room to spare) beaker, add stir bar, and set to stir (about setting 120 on stir plate). Be careful not to disturb cell pellet.

3. Add precipitation mixture to the stirring supernatant (a heavy white precipitate will form immediately) – stir for 10-15 minutes.

For each litre of cells:                         Final Concentration:
60 ml of 1M Tris pH 8                        60 mM Tris pH 8
1 ml of 5M Calcium Chloride            5mM CaCl₂
1 ml of 1M Nickel Chloride               1mM NiCl₂

4. Spin again in 1L bottles, 6000 rpm, 15 min. AT ROOM TEMPERATURE

5. Put filtered supernatant into a clean beaker with stir bar

6. Optional – Filter the supernatant through one Millipore Type HA filter with one Millipore glass fibre prefilter set on top (probably no need to do this unless you see lots of white flakes)

7. Add 2 ml resuspended Ni-NTA slurry per litre of supernatant

8. Stir (setting 120) AT ROOM TEMPERATURE for (at least) 3 hours

9. Collect the NiNTA using a Sintered glass funnel attached to a Buchner flask & vacuum.

DO NOT ALLOW Ni-NTA BEADS TO DRY

10. Wash with approx 200 ml 20 mM imidazole in 1X HBS (again directly in glass Buchner funnel)

11. Transfer washed resin to polyprep column and allow to form column. (https://www.bio-rad.com/en-uk/product/poly-prep-chromatography-columns?ID=4f7ce1ea-7c12-44e6-b435-049b88180b09)

12. Once wash buffer almost down to top of column add 200 mM imidazole in 1X HBS to elute the protein (use at least one column volume, collect fractions or entire elution as needed)

IMPORTANT!!!! DO NOT RENICKEL OVERNIGHT IN COLD ROOM.

You may recover more protein by returning the NiNTA beads from the column to the supernatant and re-incubating. However, if you do this at 4ºC you will end up with nickel beads lost in a sea of heavy white precipitate!!!! If you must re-nickel, add Na azide (3ml per litre, from 10% w/v stock)) add back the nickel beads from initial purification and stir overnight - carry out steps 4-11 again.

13. Measure the A₂₈₀ of the fractions and select those containing your protein for collection (analysis on SDS-PAGE recommended).

14. Pool relevant fractions and prepare for Gel Filtration chromatography.

15. Monomeric IL-10 and receptors should be purified using an Superdex 75 10/300 column (GE Healthcare), complexes are purified with a BioRad EnRich650 10x300 column. Sample loading should be no more than 1ml per column (0.5 ml preferred). Both pre- equilibrated with 1x HBS buffer.

Inclusion bodies

• Dimeric IL-10 is made via inclusionbodies
• Protocol is below

Inclusion body expression

Resuspension buffer: 50 mM Tris-HCl pH 8.0, 25% (w/v) sucrose, 1 mM Na EDTA, 10 mM DTT (add fresh), 0.2 mM PMSF (add fresh)

2x Lysis buffer: 100 mM Tris-HCl pH 8.0, 2% (v/v) TritonX-100, 200 mM NaCl, Benzonase 10 µL, 10 mM DTT (add fresh), 5 mM MgCl₂, 0.2 mM PMSF (add fresh)

Wash buffer 1: 50mM Tris-HCl pH 8.0, 0.5% Triton X-100, 100 mM NaCl, 1 mM Na EDTA, 1 mM DTT (add fresh), 0.2 mM PMSF (add fresh). Keep at 4ºC while using

Wash buffer 2: 50 mM Tris-HCl pH 8.0, 1 mM Na EDTA, 1 mM DTT (add fresh), 0.2 mM PMSF (add fresh). Keep at 4ºC while using

Refolding Buffer: 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 5 mM EDTA, 2 mM reduced glutathione (GSH) and 0.2 mM oxidized glutathione (GSSG). Keep at 4ºC while using

Protocol given for 1L of BL21 culture Day 1 (Transformation):

• Place cells on ice for 5-10 mins
• Add 1-2 uL of plasmid (pET vector) to cells
• Incubate on ice for 10 mins
• Heat shock at 42ºC for 42 seconds
• Incubate on ice for 2 mins
• Add the cells to 10 mLs of terrific broth/ampicillin (TB/Amp) media
• Place in 50 mL falcon tube with cap slightly ajar
• Incubate at 37ºC, shaking overnight
• Place the 1L TB/Amp media needed for tomorrow at room temp

Day 2 (Induction):

• Add the 10 mL overnight culture to 1 litre of terrific broth/ampicillin media and grow until the OD₆₀₀ reached 0.5-0.7
• Induce using 1 mM final concentration of Isopropyl β-d-1-thiogalactopyranoside (IPTG)
• Incubate at 37⁰C for 3 to 5 hours (3 minimum, 5 maximum)
• Centrifuge cultures at 6000 x g for 15 minutes and discard the supernatant
• Resuspend cell pellets in 25 mL of resuspension buffer per litre of original culture and freeze at -80ºC overnight or until needed

Day 3 (Lysis):

• Defrost cell suspension (usually place the tubes in warm (not hot!) water to speed it up)
• Add 25 mL of 2X lysis buffer (1X final concentration) to the cells that were previously resuspended in 25 ml of resuspension buffer. For 1 litre of prep you should now have 50 mls
• Incubate for 20 minutes at room temperature on a rotator.
• Add 10 mM EDTA (final concentration) to the suspension
• Divide the suspension into falcon tubes so that no tube has more than 30mLs
• Sonicate the cells (roughly 4-10 cycles of 15 seconds on/off, 15 microns per tube Soniprep150) in an ice-bath
• Centrifuge at 7000 g for 15 minutes (4⁰C), remove supernatant
• Resuspend in wash buffer 1 (50 mls per litre of original culture). I use a glass rod to break up the bulk of the pellet
• Repeat the sonication and washing step a total of three times. The solution should appear white/grey
• Resuspend the pellet after the final centrifugation step in wash buffer 2 (detergent- free), 100 mls per litre of original culture
• Centrifuge as above and remove supernatant
• The pellet contains the protein of interest as inclusion bodies
• Solubilise the pellet in 10 mL of 6M GuHCl per litre of original culturefor 30 minutes at room temperature
• Centrifuge again at 7000 g for 15 mins to clarify the solution
• Take the supernatant forward for refolding, any debris left in the tube from centrifugation can be disposed of
• Add the solubilised solution dropwise into refolding buffer (10 mL of solution into 200 mL of refolding buffer) at 4⁰C and incubate with gentle spinning overnight at 4⁰C

Day 4-10 (Dialysis):

• Precipitant will probably be present, this is normal
• Filter using a 0.45 um filterand vacuum pump (often centrifuge in conical bottlesbefore filtration to remove bulk of precipitant)
• The solution needs to be dialysedinto HBS as it will strip the Ni-NTA if used as is now
• Dialyse the 200 mLs into 2L of HBS using 14,000 Da cut off membrane (Sigma)
• Place the dialysis in the cold room with a spinner on gently overnight
• Replace with new HBS every day for 5-7 days (can leave for 2 days if needed in 4L of HBS)

Day 11-ish:

• Purify via Ni-NTA as normal except only incubate with the Ni-NTAfor 1 hour (even with dialysis the Ni-NTA still doesn't like it)
• These proteins need to be run on the Superdex 75 as sometimes we get two peaks, monomer and dimer. Varies with prep.
• Take the early peak as the dimer
• If using for cell work endotoxin removal needs to be done. This can be done the next day if proteins are stored at 4 degrees overnight

Day 12 (endotoxin removal)

• Do all steps at 4 degrees
• Make up endotoxin-free:
  • HBS
  • HBS/150 mM NaCl/20 mM imidazole/ 0.1% Triton-X114
  • HBS/20 mM imidazole
  • HBS/200 mM imidazole
  • Use a new bottle of MilliQ water, sterilefalcon tubes/insect bottlesfor solutions, new packets of filter tips, change gloves regulatory etc to reduce any chance of endotoxin contamination
  • Add 1 mL of Ni-NTA agarose to a poly-prep column
  • Wash with 10 mL of HBS
  • Add the protein to the column and collect the flow through in 0.5 mL aliquots
  • Measure the A₂₈₀ of the aliquots to ensure all protein has bound the column. Any unbound protein should be re-added to the column and check the elute until the flow through has no protein detectable
  • Wash the column with 50 column volumes (i.e., 50 mLs) of ice-cold HBS/150 mM NaCl/20 mM imidazole/ 0.1% Triton-X114 to remove endotoxin (usually need to add 10 mLs, 5 times)
  • Wash the column with 20 column volumes (2 mLs) of HBS/20 mM imidazole
  • Elute the endotoxin-free protein using 4 column volumes (4 mLs) of HBS/200 mM imidazole into a clean falcon tube
  • Buffer exchange the proteininto HBS using PD-10 columns(GE Healthcare) following the manufacturer's protocol
  • Measure the A₂₈₀ and filter sterilise the protein using the 0.2 um spintubes
  • Aliquot the protein into sterile PCR tubes
  • Snap freeze in liquid nitrogen and store at -80 degrees
  • Keep an aliquot to measure endotoxin levels (can be done the next day)

Day 13 (endotoxin level measurement)

• Use Pierce LAL Chromogenic Endotoxin Quantitation Kit (Thermo) following the manufacturer's protocol
• I usually make up a 1/2, 1/5 and 1/10 dilutionof the protein in the endotoxin-free water that they provide
• My protein endotoxin levels have always been below the level of detection of the kit

Category