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

Summary

This protocol describes a purification method for recombinant tag-free calcium binding protein S100A12 and its ion-induced oligomers for human monocyte stimulation assays.

Abstract

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for immune cell stimulations. This protocol is based on a two-step chromatography strategy, which comprises protein pre-purification on an anion-exchange chromatography column and a subsequent polishing step on a hydrophobic-interaction column. This strategy produces S100A12 protein of high purity and yield at manageable costs. For functional assays on immune cells eventual remnant endotoxin contamination requires careful monitoring and further cleaning steps to obtain endotoxin-free protein. The majority of endotoxin contaminations can be excluded by anion-exchange chromatography. To deplete residual contaminations, this protocol describes a removal step with centrifugal filters. Depending on the available ion-strength S100A12 can arrange into different homomultimers. To investigate the relationship between structure and function, this protocol further describes ion-treatment of S100A12 protein followed by chemical crosslinking to stabilize S100A12 oligomers and their subsequent separation by size-exclusion chromatography. Finally, we describe a cell-based assay that confirms the biological activity of the purified protein and confirms LPS-free preparation.

Introduction

S100A12 is a calcium binding protein which is predominantly produced by human granulocytes. The protein is overexpressed during (systemic) inflammation and its serum levels, particularly in (auto)inflammatory diseases such as systemic juvenile idiopathic arthritis (sJIA), familial Mediterranean fever (FMF) or Kawasaki disease (KD) can inform about disease activity and response to therapy. Depending on pattern recognition receptors (PRRs) such as toll-like receptors (TLRs), the innate immune system can be activated by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharides (LPS) or damage associated molecular patterns (DAMPs; also termed ‘alarmins’). DAMPs are endogenous molecules such as cellular proteins, lipids or nucleic acids¹. DAMP-functions are well described for the members of the calgranulin protein family, S100A8/A9 and S100A12², which are also reported to operate as divalent metal ion-chelating antimicrobial peptides^3,4,5,6. Depending on the available ion strength S100A12 can, like other members of the S100 family, arrange into different homomultimers and until recently the impact of S100A12-oligomerisation on PRR-interaction, particularly TLR4, was unknown.

The protein’s monomeric form (92 amino acids, 10.2 kDa) consists of two EF-hand helix-loop-helix structures connected by a flexible linker. The C-terminal EF-hand contains the classical Ca²⁺-binding motif whereas the N-terminal EF-hand exhibits an S100 protein-specific extended loop structure (‘pseudo-EF-hand’) and reveals reduced Ca²⁺-affinity. Ca²⁺-binding by S100A12 can induce a major conformational change in the proteins’ C-terminus, which results in exposure of a hydrophobic patch on each monomer and forms the dimerization interface. Thus, under physiological conditions, the smallest quaternary structure formed by S100A12 is a non-covalent dimer (approximately 21 kDa) in which individual monomers are in antiparallel orientation. When arranged as dimer, S100A12 is reported to sequester Zn²⁺ as well as other divalent metal ions, e.g., Cu²⁺ with high affinity⁷. These ions are coordinated at the S100A12 dimer interface by amino acids H15 and D25 of one subunit and H85 as well as H89 of the anti-paralleling other subunit^8,9,10. While earlier studies propose that Zn²⁺-loaded S100A12 may induce the protein’s organization into homo-tetramers (44 kDa) and to result in increased Ca²⁺-affinity^11,12, recent metal titration studies⁶ suggest Ca²⁺-binding by S100A12 to increase the protein’s affinity to Zn²⁺. Once the S100A12 EF-hands are fully occupied by Ca²⁺, additional Ca²⁺ is thought to bind between dimers, triggering hexamer formation (approximately 63 kDa). The architecture of the hexameric quarternary structure is clearly different from that of the tetramer. It is proposed that the tetramer interface is disrupted to give rise to new dimer-dimer interfaces which benefits hexamer formation¹⁰. S100A12 is almost exclusively expressed by human granulocytes where it constitutes about 5% of all cytosolic protein¹³. In its DAMP function S100A12 was historically described as agonist of the multi-ligand receptor for advanced glycation end-products (RAGE), then termed extracellular newly identified RAGE-binding protein (EN-RAGE)¹⁴. Albeit we earlier reported biochemical S100A12-binding to both RAGE and TLR4¹⁵, we recently demonstrated human monocytes to respond to S100A12 stimulation in a TLR4-dependent manner¹⁶. This requires arrangement of S100A12 into its Ca²⁺/Zn²⁺-induced hexameric quarternary structure¹⁶.

Here we describe a purification procedure for recombinant human S100A12 and its ion-induced oligomers for immune cell stimulations^16,17. This is based on a two-step chromatography strategy, which initially includes an anion-exchange column to isolate and concentrate the protein and remove bulk contaminations (e.g., endotoxins/lipopolysaccharides)¹⁸. Ion-exchange chromatography resins separate proteins on the basis of different net surface charges. For acidic proteins like S100A12 (isoelectric point of 5.81), a buffer system with a pH of 8.5 and a strong anion-exchange resin leads to a good separation. Bound proteins were eluted with a high-salt buffer gradient. With an increase of ionic strength negative ions in the elution buffer compete with proteins for charges on the surface of the resin. Proteins individually elute depending on their net charge and in result of that, the buffers described herein allow to isolate and concentrate the overexpressed S100A12 protein. Due to negatively charged groups in lipopolysaccharides, these molecules also bind to anion-exchange resins. However, their higher net charge results in later elution in the applied high-salt gradient. The second step of the purification procedure has been introduced for polishing purposes. This makes use of the calcium binding ability of S100A12 and removes remaining impurities on a hydrophobic-interaction column. Calcium binding of S100A12 leads to a conformational change and an exposure of hydrophobic patches on the surface of the protein. On that condition, S100A12 interacts with the hydrophobic surface of the resin. Upon calcium-chelating by EDTA, this interaction is reversed. In the presence of ions, especially calcium and zinc, S100A12 arranges into homomeric oligomers. To study structure-function relationships of the different oligomers, we stabilized dimeric, tetrameric and hexameric recombinant S100A12 with a chemical crosslinker and separated the complexes on a size-exclusion chromatography column. Finally, to analyze functionality and biological activity of the purified protein and its ion-induced oligomers, the cytokine release of S100A12 and LPS stimulated monocyte can be compared.

Various methods for purifying S100A12 have been described so far. Jackson et al.¹⁹, for example, published a protocol with purification via an anion-exchange column and a subsequent size-exclusion chromatography. Purification polishing on a size-exclusion column leads to good results, but―due to for example limited loading volumes―is less flexible in scalability. A different approach, published by Kiss et al.²⁰, describes purification of tagged protein via Ni²⁺ affinity column as the first purification step, followed by enzymatic cleavage to remove the tag and further purification steps. In contrast to the aforecited studies^19,20, the produced protein as described in this protocol is determined for experiments on immune cells. Therefore, remnant endotoxin contamination from bacterial culture is a challenge. Although different approaches for endotoxin removal have been described so far, there is no uniform method that works equally well for any given protein solution^21,22.

In summary, our protocol combines the advantages of a tag-free expression in a bacterial system with efficient endotoxin removal and high yield of pure protein.

Protocol

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.

Representative Results

Following pre-purification on the AIEX column (Figure 1A-C) and subsequent calcium-dependent HIC (Figure 2A,B), highly pure protein was obtained (Figure 2C). In addition, measurements of endotoxin revealed successful LPS removal. The LPS content following AIEX was measured in a 1:10 dilution above the assay detection limit, i.e., above 500 EU/mL. After the first filtration through a 50 kDa filter unit, the LPS content was reduced to 1 EU/mL. Following concentration with a 3 kDa filter unit and additional filtration through 50 kDa, the measured LPS contamination was 0.08 EU/mL.

As an additional control, human monocytes were stimulated with the produced wildtype protein (Figure 3A,B). Polymyxin B treatment abrogates TNFα release from LPS-stimulated monocytes, which cannot be observed with S100A12. On the other hand, heat-treatment of both LPS and S100A12 abolishes the protein’s capacity to stimulate cells, while this does not affect cellular response to LPS-stimulation.

Protein exposure to different ions results in arrangement of different S100A12 oligomers (Figure 4A). Chemical crosslinking allows to capture defined complexes such as dimers, tetramers, and hexamers as well as transition states (e.g., ‘trimers’, band at approximately 30 kDa). In order to induce a pronounced shift of the oligomer-equilibrium prior to crosslinking, an excess of ions was applied (Figure 4B).

Isolated oligomers in equal molar concentrations (Figure 5A-C) were then used for monocyte stimulation to compare signaling abilities via PRRs. Monocyte-stimulation with hexameric S100A12 resulted in pronounced TNFα release (Figure 6). Remnant cytokine release could be detected from cells stimulated with tetrameric S100A12, while treatment with dimeric protein does not induce TNFα release.

Figure 1: Results of anion-exchange chromatography. (A) A chromatogram with absorbance at 280 nm (A₂₈₀) and the percentage of elution buffer B (dashed line). Methods blocks are indicated with A = wash unbound sample, B = linear gradient with elution buffer (buffer B), C = wash out with buffer B, and D = re-equilibration in buffer A. (B) Focus on the relevant peaks with fraction tube numbers in red. (C) Selected fractions were analyzed on 15% Coomassie-stained SDS-PAGE. Please click here to view a larger version of this figure.

Figure 2: Results of hydrophobic-interaction chromatography. (A) A chromatogram with absorbance at 280 nm (A₂₈₀) and the percentage of elution buffer B (dashed line). Methods blocks are indicated with A = wash unbound sample, B = elution with buffer B, and C = re-equilibration in buffer A. (B) Focus on the relevant peaks with fraction tube numbers in red. (C) Analyzed fractions on 15% Coomassie-stained SDS-PAGE. Please click here to view a larger version of this figure.

Figure 3: Primary human monocytes were stimulated at indicated concentrations. LPS (A) or S100A12 (wildtype, B) were left untreated or heat-denaturated (99 °C, 10 min). Both conditions were tested in the presence and absence of polymyxin B. Please click here to view a larger version of this figure.

Figure 4: S100A12 protein was crosslinked with BS³ after incubation in HBS buffer containing 5 mM Ca²⁺ and indicated Zn²⁺ concentrations. (A) Increasing Zn²⁺ concentrations induce arrangement of S100A12 into tetramers and hexamers upon separation on 4−20% Coomassie-stained SDS-PAGE. (B) Representative result of crosslinked oligomers with conditions as used for separation on a size-exclusion column. S100A12 was crosslinked in presence of either 25 mM Ca²⁺ (lane 1) or 25 mM Ca²⁺ and 1 mM Zn²⁺ (lane 2). (S100A12)₂ = dimer; (S100A12)₄ = tetramer; (S100A12)₆ = hexamer. Please click here to view a larger version of this figure.

Figure 5: S100A12 oligomers were separated on a size-exclusion column. (A) Chromatogram of hexamer/tetramer separation after crosslinking in HBS buffer with 25 mM CaCl₂ and 1 mM ZnCl₂. (B) Chromatogram for tetramer/dimer separation in HBS buffer with 25 mM CaCl₂. (C) Example of pooled and concentrated oligomers after separation on a Coomassie-stained 4−15% gradient SDS-PAGE. Lane 1 = dimer; lane 2 = tetramer; lane 3 = hexamer. Please click here to view a larger version of this figure.

Figure 6: Stimulation of monocytes with purified S100A12 oligomers. TNFα-release after 4 h incubation was quantified by ELISA. The data show the mean value from two independent experiments. Please click here to view a larger version of this figure.

Bed Volume (CV)	75 mL
Monitor	Absorbance at 280 nM
Pressure Max	3 bar
Column buffer A	20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 8.5
Column buffer B	20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1 M NaCl, pH 8.5
Sample Volume	variable
Flow Rate	1−2 mL/min
Temperature	4 °C

Table 1: Detailed information on the applied parameters of anion-exchange chromatography.

Block	Volume	Buffer	Outlet
Equilibration	1−2 column volumes (CVs)	A	Waste
Sample load	n/a	A	Waste
Wash out unbound sample	1 CV	A	High volume outlet
Gradient–Elution	0−100 % Buffer B in 1 CV	A to B	Fraction collector
Wash out–Buffer B	1 CV	B	Waste
Re-Equilibration	2 CVs	A	Waste

Table 2: Detailed information on the used method of anion-exchange chromatography.

Bed Volume (CV)	125 mL
Monitor	Absorbance at 280 nM
Pressure Max	4 bar
Column buffer A	20 mM Tris, 140 mM NaCl, 25 mM CaCl2, pH 7.5
Column buffer B	20 mM Tris, 140 mM NaCl, 50 mM EDTA, pH 7.5
Sample Volume	variable
Flow Rate	1−2 mL/min
Temperature	4 °C

Table 3: Detailed information on the applied parameters of hydrophobic-interaction chromatography.

Block	Volume	Buffer	Outlet
Equilibration	1−2 column volumes (CVs)	A	Waste
Sample load	n/a	A	Waste
Wash out unbound sample	1−2 CVs	A	High volume outlet
Elution	100 % Buffer B	B	Fraction collector
Wash out–Buffer B	1 CV	B	Waste
Re-Equilibration	2 CVs	A	Waste

Table 4: Detailed information on the used method of hydrophobic-interaction chromatography.

Bed Volume (CV)	320 mL
Monitor	Absorbance at 280 nm
Pressure Max	3 bar
Column buffer A	20 mM Hepes, 140 mM NaCl, pH 7.2
Sample Volume	Up to 13 mL
Flow Rate	1−1.5 mL/min
Temperature	12−15 °C

Table 5: Detailed information for the applied parameters of size-exclusion chromatography.

Supplemental Table 1: Preparation of buffers and stock solutions. Please click here to download this file.

Discussion

In this protocol, we describe tag-free bacterial expression of human S100A12 and its purification as well as separation into different ion-induced oligomers for immune cell stimulation. Compared to published literature on S100A12 protein purification^8,23,24, the use of high CaCl₂ (25 mM) in hydrophobic-interaction chromatography is to our knowledge unique. Several protocols applying concentrations from 1 to 5 mM do produce pure protein, yet we observed a several times higher yield following our approach using 25 mM CaCl₂ instead. This might be explained by a hierarchy of protein interaction with the column material:S100A12 can directly bind to the column material but the excess of Ca²⁺ may also facilitate indirect binding of S100A12-dimers to the already column-bound protein⁸. Thus, high Ca²⁺ concentrations may enlarge the surface available for S100A12 purification. Elution (by using a linear gradient) of S100A12 from HIC as one early (indirectly bound S100A12) and one very late peak (column material bound protein) may support this speculation (data not shown).

For production of recombinant S100A12 (as well as other proteins) at high yields and manageable costs, protein expression in E. coli is still the method of choice. However, the inevitable contamination with bacterial endotoxins remains a problem, when proteins are determined for cell culture experiments, particularly in studies involving innate immune cells. To our experience, even commercially available proteins explicitly declared for cell culture use can contain endotoxin contaminations up to 1 EU/µg protein, which can significantly skew assays. Therefore, a complete removal of endotoxins is mandatory. Endotoxin monomers in solution range from molecular weights of 10 to 20 kDa, but they can form micelles and structures with higher molecular weights. The formation of very large structures is, for example, promoted through bivalent ions^21,25.

According to our protocol, we verify the endotoxin-free production of S100A12 protein by combining high-sensitivity endotoxin measurements with monocyte stimulation assays. We consider such combination particularly meaningful as a) low-level endotoxin contamination may be difficult to assess depending on the sensitivity of the assay and b) the use of polymyxin B as LPS inhibitor on monocytes may result in difficult to interpret data due to exclusive polymyxin B effects on cells^26,27. Polymyxin B as well as other cationic peptides are reported to bind LPS via negatively charged lipid A²⁸. As the solvent exposed surface of S100A12 also contains large negatively charged patches the observed reduction of TNFα-release from S100A12-stimulated human monocytes in presence of polymyxin B (Figure 3B) may be due to a) unspecific direct binding of polymyxin B to S100A12 and/or b) direct effects of polymyxin B on stimulated cells^26,27. Due to the known limitations of both the detection of low-level endotoxin contamination as well as unspecific polymyxin B effects, our protocol further contains a heat-inactivation step to clearly distinguish between LPS- and protein-mediated TLR4-signaling.

Use of LPS-free S100A12 for generation and purification of defined ion-induced oligomers is critical and extra attention should be paid to their subsequent purification to avoid eventual re-introduction of endotoxin via buffers or column material and thus further protein-demanding LPS-depletion via endotoxin removal resins.

The relevance of oligomerization for the biological function of proteins can be assessed by different means. In case of S100A12, we used surface plasmon resonance as well as targeted amino acid exchanges at ion-binding sites and―to most precisely define the protein-complex able to bind and signal through TLR4―we employed chemical crosslinking of Ca²⁺/Zn²⁺-pulsed recombinant S100A12¹⁶. Chemical crosslinking of S100A12 under different ionic conditions snap-freezes a momentary state including several oligomeric forms that are in transition. From ion titration experiments, we defined conditions under which dimeric, tetrameric or hexameric oligomers could be determined as the predominant oligomers¹⁶. In addition, previous experiments have shown that an excess of ions is beneficial for comparable, stable crosslinking and subsequent purification, although oligomerization can also be induced at significantly lower ion concentrations. However, purifying these oligomers by size-exclusion chromatography results in good, but not absolute separation. Still, the selective enrichment of oligomers allows for reliable downstream analyses.

In summary, this protocol provides a method for purification of LPS-free human S100A12 or related calcium binding proteins. To fix ion-induced conformational changes, chemical crosslinking and subsequent complex separation by size-exclusion chromatography is a useful tool to understand the relevance of protein oligomerization for downstream biological processes.

Materials

Name	Company	Catalog Number	Comments
pET11b vector	Novagen
BL21(DE3) competent E. coli	New England Biolabs	C2527
100x Non-essential amino acids	Merck	K 0293
25% HCl	Carl Roth	X897.1
4−20% Mini-PROTEAN TGX Protein Gels	BioRad	4561093
Ampicillin sodium salt	Carl Roth	HP62.1
BS3 (bis(sulfosuccinimidyl)suberate) - 50 mg	ThermoFisher Scientific	21580
Calciumchlorid Dihydrat	Carl Roth	5239.1
Coomassie Briliant Blue R250 Destaining Solution	BioRad	1610438
Coomassie Briliant Blue R250 Staining Solution	BioRad	1610436
EasySep Human Monocyte Enrichment Kit	Stemcell	19059	Magnetic negative cell isolation kit
EDTA disodium salt dihydrate	Carl Roth	8043.1
EGTA	Carl Roth	3054.3
EndoLISA	Hyglos	609033	Endotoxin detection assay
Endotoxin-Free Ultra Pure Water	Sigma-Aldrich	TMS-011-A	Ultrapure water for preparation of endotoxin-free buffers
EndoTrap red	Hyglos	321063	Endotoxin removal resin
FBS (heat-inactivated)	Gibco	10270
HBSS, no calcium, no magnesium	ThermoFisher Scientific	14175053
Hepes	Carl Roth	9105
Hepes (high quality, endotoxin testet)	Sigma-Aldrich	H4034
hTNF-alpha - OptEia ELISA Set	BD	555212
IPTG (isopropyl-ß-D-thiogalactopyranosid)	Carl Roth	CN08.1
L-Glutamine (200 mM)	Merck	K 0282
LB-Medium	Carl Roth	X968.1
Lipopolysaccharides from E. coli O55:B5	Merck	L6529
Pancoll, human	PAN Biotech	P04-60500	Separation solution (density gradient centrifugation)
Penicillin/Streptomycin (10.000 U/mL)	Merck	A 2212
Phenyl Sepharose High Performance	GE Healthcare	17-1082-01	Resin for hydrophobic interaction chromatography
Polymyxin B	Invivogen	tlrl-pmb
Protease inhibitor tablets	Roche	11873580001
Q Sepharose Fast Flow	GE Healthcare	17-0510-01	Resin for anion-exchange chromatography
RoboSep buffer	Stemcell	20104	Cell separation buffer (section 5.1.4)
RPMI 1640 Medium	Merck	F 1215
Sodium chloride (NaCl)	Carl Roth	3957.2
Sodium hydroxide	Carl Roth	P031.1
Tris Base	Carl Roth	4855.3
Zinc chloride	Carl Roth	T887
Labware
0,45 µm syringe filter	Merck	SLHA033SS
14 mL roundbottom tubes	BD	352059
2 L Erlenmyer flask	Carl Roth	LY98.1
24 well suspension plates	Greiner	662102
5 L measuring beaker	Carl Roth	CKN3.1
50 mL conical centrifuge tubes	Corning	430829
50 mL high-speed centrifuge tubes	Eppendorf	3,01,22,178
Amicon Ultra-15 Centrifugal Filter Unit MWCO 3 kDa	Merck	UFC900324
Amicon Ultra-15 Centrifugal Filter Unit MWCO 50 kDa	Merck	UFC905024
Culture dish (100 mm)	Sarstedt	83.3902
Dialysis Tubing Closures	Spectrum	132738
EasySep magnet 'The Big Easy`	Stemcell	18001
Fraction collector tubes 5 mL	Greiner	115101
Lumox film, 25 µm, 305 mm x 40 m	Sarstedt	94,60,77,316	Film for monocyte culture plates
Spectra/Por Dialysis Membrane (3.5 kDa)	Spectrum	132724
Steritop filter unit	Merck	SCGPT01RE
Equipment
37 °C Incubator (with shaking)	New Brunswick Scientific	Innova 42
ÄKTA purifier UPC 10	GE Healthcare		FPLC System
Fraction collector	GE Healthcare	Frac-920
Centrifuge (with rotor A-4-81)	Eppendorf	5810R
Fixed angle rotor	Eppendorf	F-34-6-38
Mini Protean Tetra Cell	BioRad	1658000EDU
NanoPhotometer	Implen	P330
Sonicator	Brandelin	UW2070
Fluorescence reader	Tecan	infinite M200PRO
pH meter	Knick	765