Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation

NOTE: Please refer to Supplemental Table 1 for preparation of buffers and stock solutions.

1. Protein expression in E. coli

1. Cloning
   1. Clone tag-free human S100A12 (NCBI Reference Sequence: NP_005612.1) into bacterial expression vector pET11b. To express the protein, transform the construct into E. coli BL21(DE3).
2. Culture
   1. Prepare a starter culture by inoculating a single colony in 5 mL of growth medium (LB broth with 100 µg/mL ampicillin) in a 14 mL round-bottom tube. Incubate overnight at 37 °C with shaking at 220 rpm. Transfer 2−4 mL of overnight culture into 400 mL of growth medium in a 2 L Erlenmayer flask and incubate the culture at 37 °C with shaking at 220 rpm.
      NOTE: Initial density of the main culture should be optical density at 600 nm (OD₆₀₀) = 0.1.
   2. Monitor the OD₆₀₀ during growth. Induce protein expression by addition of 1 M isopropyl-ß-D-thiogalactopyranosid (IPTG) to a final concentration of 1 mM at OD₆₀₀ = 0.5−0.6. Incubate at 37 °C and 220 rpm for additional 4 h.
      NOTE: In general, an OD₆₀₀ of 0.6 will be reached after 1.5−2.5 h at 37 °C.
   3. Prepare 50 mL sonication buffer by dissolving 50 mM Tris, 50 mM NaCl and 1 mM ethylenediamine tetraacetic acid (EDTA) in 40 mL of deionized water. Adjust pH with HCl to 8.0 and make up to 50 mL. Add protease inhibitor (1 tablet per 50 mL solution) and equilibrate the buffer to 4 °C.
   4. Transfer the bacterial culture into suitable centrifuge bottles and harvest the cells at 3,200 x g for 30 min at 4 °C. Discard the supernatant and resuspend the pellet in 25 mL of ice-cold sonication buffer. Henceforth keep the cells on ice.
      NOTE: Resuspended cells can be stored at -20 °C for short-term and at -80 °C for long-term.
3. Sonication/lysis
   1. Sonicate the cells for 6 cycles of 30 s on ice. After each cycle, rest cells for 30−60 s to protect the cells from overheating.
   2. Transfer the cell suspension to a pre-chilled 50 mL high-speed centrifugation tube and centrifuge in a fixed angle rotor at 15,000 x g for 30 min at 4 °C. Decant the cleared lysate which contains the soluble cytosolic proteins into a fresh 50 mL tube and discard the pellet.

2. Protein Purification

1. Anion-exchange chromatography
   1. Dialysis
      1. Prepare anion-exchange chromatography (AIEX) buffer A by dissolving 20 mM Tris, 1 mM EDTA and 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’-tetraacetic acid (EGTA) in deionized water and adjust the pH to 8.5 with HCl. For dialysis prepare 2 x 5 L and for chromatography 2x 1 L of AIEX buffer A.
         NOTE: The dialysate volume should be at about 100 times the sample volume. All buffers used for chromatography should be filtered (0.45 µm or smaller) and degassed (e.g., by ultrasonic bath or vacuum degassing).
      2. Cut dialysis tubing (molecular weight cut-off [MWCO]: 3.5 kDa) into an appropriate length with additional space for air to ensure sample buoyancy above the rotating stir bar.
         NOTE: Glycerol preserves the membrane and must be removed before use.
      3. To reduce the viscosity of the cleared protein solution from step 1.3.2, dilute the solution with 25 mL of AIEX buffer A to facilitate subsequent application to the chromatography column. Attach the first closure onto the tubing, load the sample into the membrane and attach the second closure at least 1 cm from the top end of the tubing.
      4. Place the 5 L container with AIEX buffer A on a stir plate, add a stir bar and the membrane filled with protein solution. Adjust the speed to rotate the sample by avoiding interference with the rotating stir-bar. Dialyze for 12−24 h at 4 °C, then replace the dialysate buffer (AIEX buffer A) by a fresh pre-cooled preparation and continue for at least 4 additional hours. Transfer the dialyzed protein solution to a 50 mL tube and filter through a 0.45 µm filter unit.
         NOTE: Storage possible.
   2. Chromatography
   3. Start the liquid chromatography system (FPLC) with general maintenance, connect column buffers AIEX A and AIEX B (AIEX buffer A with 1 M NaCl) and the anion-exchange resin containing column. Refer to Table 1 for general chromatographic parameters.
      NOTE: Buffers, column and FPLC equipment should be equilibrated to the same temperature before starting the run (refer to chromatographic parameters in Table 1, Table 2, Table 3, Table 4, and Table 5).
   4. Equilibrate the column with AIEX buffer A, subsequently load the sample onto the column and elute the proteins with a linear gradient from 0% to 100% high-salt buffer (AIEX B). Refer to Table 2 for a detailed method protocol.
   5. Collect 2 mL fractions during elution and analyze 10 µL of each fraction on a Coomassie-stained 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Pool the fractions containing S100A12 protein for dialysis.
      NOTE: The molecular weight of S100A12 is 10,575 Da.
2. Calcium-dependent hydrophobic-interaction chromatography (HIC)
   1. Dialysis
      1. Dialyze the protein solution against 20 mM Tris, 140 mM NaCl, pH 7.5 following the procedure described in section 2.1.1.
   2. Chromatography
      1. Prepare 1 L of chromatography buffer HIC A by dissolving 20 mM Tris, 140 mM NaCl and 25 mM CaCl₂ in deionized water and adjust the pH to 7.5. For HIC buffer B, dissolve 20 mM Tris, 140 mM NaCl and 50 mM EDTA. Adjust the pH to 7.0 and filter and degas the buffers. Add CaCl₂ to the sample to a final concentration of 25 mM and filter through 0.45 µm. Equilibrate HIC buffers and sample to 4 °C (column temperature).
      2. Start the liquid chromatography system with general maintenance, connect column buffers HIC A and B and the column. Refer to Table 3 for further chromatographic parameters.
      3. Equilibrate the column, load the sample and extend the ‘wash unbound sample’ block until the UV signal reaches baseline level again. Then start elution with a calcium chelator containing buffer (EDTA). Refer to Table 4 for a detailed method protocol.
         NOTE: Previous experiments have shown that an excess of calcium seems to be beneficial for binding of S100A12 to the chromatography resin.
      4. Collect peak fractions of 2 mL and analyze 10 µL of each fraction on a Coomassie-stained 15% SDS-PAGE. Pool pure S100A12 fractions and dialyze against Hepes-buffered saline (HBS; 20 mM Hepes, 140 mM NaCl, pH 7.0) as described in section 2.1.1.
         NOTE: Extinction coefficient of monomeric S100A12 is 2980 M^-1 cm^-1.

3. Detection and Removal of Endotoxin

1. Detection of endotoxin
   1. To determine the endotoxin contamination, measure concentrations of diluted protein from step 2.2.2.4. (e.g., 1:10 and 1:100 in HBS) using an enzyme-linked immunosorbent assay (ELISA)-based, fluorescent endotoxin detection assay (Table of Materials). Perform this assay by following the manufacturer’s protocol.
      NOTE: Use freshly prepared HBS solutions dissolved in ultrapure deionized water to avoid (new) endotoxin contamination by the buffer.
2. Removal of endotoxin and concentration of protein
   1. Load 15 mL of sample onto a 50 kDa centrifugal filter unit and centrifuge at 3,200 x g and 10 °C for approximately 10 min. Transfer the flow-through into a fresh vessel (on ice) and refill and centrifuge the 50 kDa filter tube as often as necessary. Wash the filter membrane twice with HBS to recover as much protein as possible after each step.
   2. Concentrate the S100A12-containing flow-through by using a 3 kDa centrifugal filter until the volume is reduced to one fifth up to one tenth of the initial loading volume (centrifugation at 3,200 x g, 10 °C for approximately 30 min). Refill the filter as often as necessary, rinse the membrane and transfer the concentrated solution to a new tube after each refill. Discard the flow-through. Filter again through 50 kDa as described above.
      NOTE: During this procedure, the loss of protein is remarkable (up to 50%), but the remaining protein preparation is completely depleted from LPS. This method yields about 10−15 mg protein from 400 mL culture.
   3. Adjust the protein solution to 1 mg/mL with endotoxin-free HBS and measure the LPS content as described in step 3.1.1. In case the protein solution is still not tested as LPS-free (<0.1 EU/mL), eliminate remnant contaminations by using an endotoxin removal resin.
      NOTE: With a protein concentration of 1 mg/mL, contamination of 0.1 EU/mL LPS equals approximately 0.01 pg LPS/µg protein.

4. Chemical Crosslinking and Oligomer Separation

1. Chemical crosslinking
   1. Prepare highly pure (endotoxin-free) stock solutions of 1 M CaCl₂ and 100 mM ZnCl₂ in ultrapure deionized water (Table of Materials). Use this buffer, freshly made, for the next step.
   2. Incubate 10 mL of purified endotoxin-free S100A12 (concentration 1 mg/mL in HBS) for 30 min at room temperature (RT) with either 25 mM CaCl₂ for dimeric/tetrameric, or 25 mM CaCl₂ and 1 mM ZnCl₂ for hexameric/tetrameric S100A12 oligomers.
   3. Prepare crosslinker by dissolving 8 mg of BS³ in 500 µL of endotoxin-free water directly before use (8 mg crosslinker for 10 mL ion-spiked protein solution equals a final concentration of 1.4 mM). Mix crosslinker and sample by pipetting and incubate for additional 30 min at RT. Quench the reaction by adding 1 M Tris-HCl, pH 7.5 to a final concentration of 50 mM and filter through 0.45 µm.
2. Size-exclusion chromatography
   1. Equilibrate the crosslinked sample to 12−15 °C (column temperature) and start the liquid chromatography system with general maintenance. Connect column buffer (HBS) and the size-exclusion column. Refer to Table 5 for detailed information.
   2. Equilibrate the column in HBS, load sample and collect peak fractions (1−2 mL) during the run. Analyze these fractions on a 4−20% gradient SDS-PAGE and pool fractions with major bands of the desired protein complex.
      NOTE: Hydrolysis of NHS ester reagents like BS³ in aqueous solutions results in a strong absorbance at 280 nm. Unbound crosslinker (molecular weight: 572 g/mol) elutes at the end of the run and results in a strong peak.
   3. Concentrate the solutions by using centrifugal filter units with MWCOs of 10 kDa (dimer), 30 kDa (tetramer) or 50 kDa (hexamer). Determine the endotoxin contamination as described in section 3.1. If necessary, remove remaining LPS with an endotoxin removal resin following the manufacturer’s recommendations (Table of Materials).

5. Functional Testing on Monocytes

1. Preparation of monocytes
   1. Isolate monocytes from human buffy coats by density gradient centrifugation and subsequent monocyte enrichment by using a magnetic bead separation kit (Table of Materials).
      NOTE: This protocol will result in approximately 5−7 x 10⁷ monocytes (one buffy coat) with a purity of 83−95%. Since the number, but also the responsiveness of cells depends strongly on the donor, the protocol may have to be scaled up (depending on the required cell count).
   2. For density centrifugation, equilibrate the separation solution (density = 1.077 g/mL) to RT and transfer 20 mL into 50 mL centrifuge tubes (2 tubes per buffy coat). Dilute blood from the human buffy coat with Hank’s buffered salt solution (HBSS) to a total volume of 60 mL and layer 30 mL of this mixture carefully on top of the separation medium. Centrifuge at 550 x g for 35 min at RT. Disable the centrifuge brake.
   3. After centrifugation, the mononuclear peripheral blood cells (PBMCs) are located directly on top of the separation medium. Transfer these cells into a fresh 50 mL centrifuge tube, make up to 50 mL with HBSS, and centrifuge at 170 x g for 10 min. Aspirate the supernatant and resuspend the cell pellet in a small volume of HBSS by pipetting.
   4. Fill the tube up to 50 mL and centrifuge at 290 x g for 10 min. Aspirate the supernatant again, resuspend the cells in HBSS (50 mL) and centrifuge at 170 x g for 10 min. Count the cells and resuspend them in cell separation buffer (Table of Materials) to a concentration of 5 x 10⁷ cells/mL.
      NOTE: Instead of HBSS, phosphate-buffered saline (PBS) can be used for washing the cells.
   5. For monocyte isolation from PBMCs, use a magnetic negative cell isolation kit and follow the manufacturer’s protocol. Count monocytes and resuspend in monocyte medium (RPMI 1640, 15% heat-inactivated fetal calf serum [FCS], 4 mM L-glutamine, 100 U/mL penicillin/streptomycin) to a concentration of 2 x 10⁶ cells/mL.
   6. To culture monocytes, coat culture dishes (e.g., 100 mm) with a hydrophobic, gas-permeable film, suitable for suspension cells (Table of Materials). Sterilize the plates by using UV light for approximately 30 min. Transfer the cells to these culture plates and let them rest over-night at 37 °C and 5% CO₂.
      NOTE: Use 15−25 mL of cell suspension per coated dish.
2. Monocyte stimulation
   1. Stimulation with S100A12 (wildtype)
      NOTE: To distinguish untreated S100A12 (end-product from section 2.2.2) from crosslinked protein, S100A12 in the following is referred to as ‘wildtype’.
      1. Transfer the rested cells into a 50 mL centrifugal tube and centrifuge at 350 x g for 10 min. Aspirate the supernatant and resuspend the cell pellet in stimulation medium (RPMI 1640, 5% heat-inactivated FCS, 4 mM L-glutamine, 100 U/mL penicillin/streptomycin) at a concentration of 2 x 10⁶ cells/mL.
      2. For stimulation, use 24 well suspension plates and add 250 µL of cell suspension per well (0.5 x 10⁶ cells/well). Add 50 µg/mL polymyxin B to the intended wells, followed by either LPS in different concentrations (25, 50, 100 and 200 pg/mL) or wildtype S100A12 (10, 20, 40, 60 µg/mL). Further, apply the protein either untreated or heat-denatured (99 °C, 10 min) in different concentrations to the cells.
         NOTE: A short heat treatment denatures S100A12 protein but has less to no effect on LPS.
      3. Incubate plates for 4 h at 37 °C and 5% CO₂. Harvest the cells by transferring the cell suspension of each well to 1.5 mL reaction tubes. Centrifuge at 500 x g for 10 min. Transfer the supernatants to fresh tubes and measure TNFα release in different dilutions (e.g., 1:2, 1:5, 1:10) with a human TNFα ELISA kit following the manufacturer’s recommendations.
   2. Stimulation with S100A12 oligomers
      1. Prepare and seed out monocytes in 24 well suspension plates as described above. Stimulate cells by adding S100A12 oligomers from step 4.2.3. in different molar concentrations (125 nM, 250 nM, 500 nM, 1000 nM).
         NOTE: In order to compare the abilities of the different oligomers to stimulate monocytes, oligomers were applied to the cells in comparable molar concentrations.
      2. Incubate for 4 h at 37 °C and 5% CO₂, harvest the cells and measure TNFα release in the supernatants as described above.