This study was supported by grants from the intramural innovative medical research program of Muenster University medical faculty (KE121201 to C.K.) and the German Research Foundation (DFG, Fo354/3-1 to D.F.).

NOTE: Please refer to Supplemental Table 1 for preparation of buffers and stock solutions.
1. Protein expression in E. coli
<ol>
	<li>Cloning
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Culture
	<ol>
		<li>Prepare a starter culture by inoculating a single colony in 5 mL of growth medium (LB broth with 100 &#181;g/mL ampicillin) in a 14 mL round-bottom tube. Incubate overnight at 37 &#176;C with shaking at 220 rpm. Transfer 2&#8722;4 mL of overnight culture into 400 mL of growth medium in a 2 L Erlenmayer flask and incubate the culture at 37 &#176;C with shaking at 220 rpm. 
		NOTE: Initial density of the main culture should be optical density at 600 nm (OD600) = 0.1.</li>
		<li>Monitor the OD600 during growth. Induce protein expression by addition of 1 M isopropyl-&#223;-D-thiogalactopyranosid (IPTG) to a final concentration of 1 mM at OD600 = 0.5&#8722;0.6. Incubate at 37 &#176;C and 220 rpm for additional 4 h. 
		NOTE: In general, an OD600 of 0.6 will be reached after 1.5&#8722;2.5 h at 37 &#176;C.</li>
		<li>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 &#176;C.</li>
		<li>Transfer the bacterial culture into suitable centrifuge bottles and harvest the cells at 3,200 x g for 30 min at 4 &#176;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 &#176;C for short-term and at -80 &#176;C for long-term.</li>
	</ol>
	</li>
	<li>Sonication/lysis
	<ol>
		<li>Sonicate the cells for 6 cycles of 30 s on ice. After each cycle, rest cells for 30&#8722;60 s to protect the cells from overheating.</li>
		<li>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 &#176;C. Decant the cleared lysate which contains the soluble cytosolic proteins into a fresh 50 mL tube and discard the pellet.</li>
	</ol>
	</li>
</ol>
2. Protein Purification
<ol>
	<li>Anion-exchange chromatography
	<ol>
		<li>Dialysis
		<ol>
			<li>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&#8217;,N&#8217;-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 &#181;m or smaller) and degassed (e.g., by ultrasonic bath or vacuum degassing).</li>
			<li>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.</li>
			<li>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&#160;cm from the top end of the tubing.</li>
			<li>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&#8722;24 h at 4 &#176;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 &#181;m filter unit. 
			NOTE: Storage possible.</li>
		</ol>
		</li>
		<li>Chromatography</li>
		<li>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).</li>
		<li>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.</li>
		<li>Collect 2 mL fractions during elution and analyze 10 &#181;L of each fraction on a Coomassie-stained 15% sodium dodecyl sulfate polyacrylamide&#160;gel&#160;electrophoresis (SDS-PAGE). Pool the fractions containing S100A12 protein for dialysis. 
		NOTE: The molecular weight of S100A12 is 10,575 Da.</li>
	</ol>
	</li>
	<li>Calcium-dependent hydrophobic-interaction chromatography (HIC)
	<ol>
		<li>Dialysis
		<ol>
			<li>Dialyze the protein solution against 20 mM Tris, 140 mM NaCl, pH 7.5 following the procedure described in section 2.1.1.</li>
		</ol>
		</li>
		<li>Chromatography
		<ol>
			<li>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 &#181;m. Equilibrate HIC buffers and sample to 4 &#176;C (column temperature).</li>
			<li>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.</li>
			<li>Equilibrate the column, load the sample and extend the &#8216;wash unbound sample&#8217; 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.</li>
			<li>Collect peak fractions of 2 mL and analyze 10 &#181;L 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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Detection and Removal of Endotoxin
<ol>
	<li>Detection of endotoxin
	<ol>
		<li>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&#8217;s protocol. 
		NOTE: Use freshly prepared HBS solutions dissolved in ultrapure deionized water to avoid (new) endotoxin contamination by the buffer.</li>
	</ol>
	</li>
	<li>Removal of endotoxin and concentration of protein
	<ol>
		<li>Load 15 mL of sample onto a 50 kDa centrifugal filter unit and centrifuge at 3,200 x g and 10 &#176;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.</li>
		<li>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 &#176;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&#8722;15 mg protein from 400 mL culture.</li>
		<li>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 (&#60;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/&#181;g protein.</li>
	</ol>
	</li>
</ol>
4. Chemical Crosslinking and Oligomer Separation
<ol>
	<li>Chemical crosslinking
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Prepare crosslinker by dissolving 8 mg of BS3 in 500 &#181;L 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 &#181;m.</li>
	</ol>
	</li>
	<li>Size-exclusion chromatography
	<ol>
		<li>Equilibrate the crosslinked sample to 12&#8722;15 &#176;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.</li>
		<li>Equilibrate the column in HBS, load sample and collect peak fractions (1&#8722;2 mL) during the run. Analyze these fractions on a 4&#8722;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.</li>
		<li>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&#8217;s recommendations (Table of Materials).</li>
	</ol>
	</li>
</ol>
5. Functional Testing on Monocytes
<ol>
	<li>Preparation of monocytes
	<ol>
		<li>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&#8722;7 x 107 monocytes (one buffy coat) with a purity of 83&#8722;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).</li>
		<li>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&#8217;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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>For monocyte isolation from PBMCs, use a magnetic negative cell isolation kit and follow the manufacturer&#8217;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.</li>
		<li>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 &#176;C and 5% CO2. 
		NOTE: Use 15&#8722;25 mL of cell suspension per coated dish.</li>
	</ol>
	</li>
	<li>Monocyte stimulation
	<ol>
		<li>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 &#8216;wildtype&#8217;.
		<ol>
			<li>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.</li>
			<li>For stimulation, use 24 well suspension plates and add 250 &#181;L of cell suspension per well (0.5 x 106 cells/well). Add 50 &#181;g/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 &#181;g/mL). Further, apply the protein either untreated or heat-denatured (99 &#176;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.</li>
			<li>Incubate plates for 4 h at 37 &#176;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 TNF&#945; release in different dilutions (e.g., 1:2, 1:5, 1:10) with a human TNF&#945; ELISA kit following the manufacturer&#8217;s recommendations.</li>
		</ol>
		</li>
		<li>Stimulation with S100A12 oligomers
		<ol>
			<li>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.</li>
			<li>Incubate for 4 h at 37 &#176;C and 5% CO2, harvest the cells and measure TNF&#945; release in the supernatants as described above.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+partial+characterization+of+canine+S100A12.">Purification and partial characterization of canine S100A12.</a> Biochimie. 92 (12), 1914-1922 (2010).</li><li>Hung, K. W., Hsu, C. C., Yu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solution+structure+of+human+Ca2+-bound+S100A12.">Solution structure of human Ca2+-bound S100A12.</a> Journal of Biomolecular NMR. 57 (3), 313-318 (2013).</li><li>Endotoxin Removal. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Application Note - Sartorius Stedim Biotech. , (2010).</li><li>Hogasen, A. K. M., Abrahamsen, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymyxin-B+Stimulates+Production+of+Complement+Components+and+Cytokines+in+Human+Monocytes.">Polymyxin-B Stimulates Production of Complement Components and Cytokines in Human Monocytes.</a> Antimicrobial Agents and Chemotherapy. 39 (2), 529-532 (1995).</li><li>Valentinis, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+effects+of+polymyxin+B+on+human+dendritic+cells+maturation+-+The+role+of+I+kappa+B-alpha/NF-kappa+B+and+ERK1/2+pathways+and+adhesion.">Direct effects of polymyxin B on human dendritic cells maturation - The role of I kappa B-alpha/NF-kappa B and ERK1/2 pathways and adhesion.</a> Journal of Biological Chemistry. 280 (14), 14264-14271 (2005).</li><li>Teuber, M., Miller, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Binding+of+Polymyxin-B+to+Negatively+Charged+Lipid+Monolayers.">Selective Binding of Polymyxin-B to Negatively Charged Lipid Monolayers.</a> Biochimica Et Biophysica Acta. 467 (3), 280-289 (1977).</li></ol>

The authors have nothing to disclose.

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 purification8,23,24, 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/&#181;g 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 ions21,25.
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 cells26,27. 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 TNF&#945;-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 cells26,27. 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&#8213;to most precisely define the protein-complex able to bind and signal through TLR4&#8213;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.

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 &#8216;alarmins&#8217;). 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 peptides3,4,5,6. 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&#8217;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 (&#8216;pseudo-EF-hand&#8217;) and reveals reduced Ca2+-affinity. Ca2+-binding by S100A12 can induce a major conformational change in the proteins&#8217; 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 subunit8,9,10. While earlier studies propose that Zn2+-loaded S100A12 may induce the protein&#8217;s organization into homo-tetramers (44 kDa) and to result in increased Ca2+-affinity11,12, recent metal titration studies6 suggest Ca2+-binding by S100A12 to increase the protein&#8217;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 stimulations16,17. 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&#8213;due to for example limited loading volumes&#8213;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 studies19,20, 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 solution21,22.
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.

<table><tbody><tr><td>pET11b vector</td><td>Novagen</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>BL21(DE3) competent &lt;em&gt;E. coli&lt;/em&gt;</td><td>New England Biolabs</td><td>C2527</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td><td>&lt;strong&gt;&amp;nbsp;&lt;/strong&gt;</td></tr><tr><td>100x Non-essential amino acids</td><td>Merck</td><td>K 0293</td><td></td></tr><tr><td>25% HCl</td><td>Carl Roth</td><td>X897.1</td><td></td></tr><tr><td>4&amp;minus;20% Mini-PROTEAN TGX Protein Gels</td><td>BioRad</td><td>4561093</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Carl Roth</td><td>HP62.1</td><td></td></tr><tr><td>BS3 (bis(sulfosuccinimidyl)suberate) - 50 mg</td><td>ThermoFisher Scientific</td><td>21580</td><td></td></tr><tr><td>Calciumchlorid Dihydrat</td><td>Carl Roth</td><td>5239.1</td><td></td></tr><tr><td>Coomassie Briliant Blue R250 Destaining Solution</td><td>BioRad</td><td>1610438</td><td></td></tr><tr><td>Coomassie Briliant Blue R250 Staining Solution</td><td>BioRad</td><td>1610436</td><td></td></tr><tr><td>EasySep Human Monocyte Enrichment Kit</td><td>Stemcell</td><td>19059</td><td>Magnetic negative cell isolation kit</td></tr><tr><td>EDTA disodium salt dihydrate</td><td>Carl Roth</td><td>8043.1</td><td></td></tr><tr><td>EGTA</td><td>Carl Roth</td><td>3054.3</td><td></td></tr><tr><td>EndoLISA</td><td>Hyglos</td><td>609033</td><td>Endotoxin detection assay</td></tr><tr><td>Endotoxin-Free Ultra Pure Water</td><td>Sigma-Aldrich</td><td>TMS-011-A</td><td>Ultrapure water for preparation of endotoxin-free buffers</td></tr><tr><td>EndoTrap red</td><td>Hyglos</td><td>321063</td><td>Endotoxin removal resin</td></tr><tr><td>FBS (heat-inactivated)</td><td>Gibco</td><td>10270</td><td></td></tr><tr><td>HBSS, no calcium, no magnesium</td><td>ThermoFisher Scientific</td><td>14175053</td><td></td></tr><tr><td>Hepes</td><td>Carl Roth</td><td>9105</td><td></td></tr><tr><td>Hepes (high quality, endotoxin testet)</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>hTNF-alpha - OptEia ELISA Set</td><td>BD</td><td>555212</td><td></td></tr><tr><td>IPTG (isopropyl-&amp;szlig;-D-thiogalactopyranosid)</td><td>Carl Roth</td><td>CN08.1</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Merck</td><td>K 0282</td><td></td></tr><tr><td>LB-Medium</td><td>Carl Roth</td><td>X968.1</td><td></td></tr><tr><td>Lipopolysaccharides from &lt;em&gt;E. coli&lt;/em&gt; O55:B5</td><td>Merck</td><td>L6529</td><td></td></tr><tr><td>Pancoll, human</td><td>PAN Biotech</td><td>P04-60500</td><td>Separation solution (density gradient centrifugation)</td></tr><tr><td>Penicillin/Streptomycin (10.000 U/mL)</td><td>Merck</td><td>A 2212</td><td></td></tr><tr><td>Phenyl Sepharose High Performance</td><td>GE Healthcare</td><td>17-1082-01</td><td>Resin for hydrophobic interaction chromatography</td></tr><tr><td>Polymyxin B</td><td>Invivogen</td><td>tlrl-pmb</td><td></td></tr><tr><td>Protease inhibitor tablets</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Q Sepharose Fast Flow</td><td>GE Healthcare</td><td>17-0510-01</td><td>Resin for anion-exchange chromatography</td></tr><tr><td>RoboSep buffer</td><td>Stemcell</td><td>20104</td><td>Cell separation buffer (section 5.1.4)</td></tr><tr><td>RPMI 1640 Medium</td><td>Merck</td><td>F 1215</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Carl Roth</td><td>3957.2</td><td></td></tr><tr><td>Sodium hydroxide</td><td>Carl Roth</td><td>P031.1</td><td></td></tr><tr><td>Tris Base</td><td>Carl Roth</td><td>4855.3</td><td></td></tr><tr><td>Zinc chloride</td><td>Carl Roth</td><td>T887</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Labware&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0,45 &amp;micro;m syringe filter</td><td>Merck</td><td>SLHA033SS</td><td></td></tr><tr><td>14 mL roundbottom tubes</td><td>BD</td><td>352059</td><td></td></tr><tr><td>2 L Erlenmyer flask</td><td>Carl Roth</td><td>LY98.1</td><td></td></tr><tr><td>24 well suspension plates</td><td>Greiner</td><td>662102</td><td></td></tr><tr><td>5 L measuring beaker</td><td>Carl Roth</td><td>CKN3.1</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>50 mL high-speed centrifuge tubes</td><td>Eppendorf</td><td>3,01,22,178</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit MWCO 3 kDa</td><td>Merck</td><td>UFC900324</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit MWCO 50 kDa</td><td>Merck</td><td>UFC905024</td><td></td></tr><tr><td>Culture dish (100 mm)</td><td>Sarstedt</td><td>83.3902</td><td></td></tr><tr><td>Dialysis Tubing Closures</td><td>Spectrum</td><td>132738</td><td></td></tr><tr><td>EasySep magnet &#039;The Big Easy`</td><td>Stemcell</td><td>18001</td><td></td></tr><tr><td>Fraction collector tubes 5 mL</td><td>Greiner</td><td>115101</td><td></td></tr><tr><td>Lumox film, 25 &amp;micro;m, 305 mm x 40 m</td><td>Sarstedt</td><td>94,60,77,316</td><td>Film for monocyte culture plates</td></tr><tr><td>Spectra/Por Dialysis Membrane (3.5 kDa)</td><td>Spectrum</td><td>132724</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck</td><td>SCGPT01RE</td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>37 &amp;deg;C Incubator (with shaking)</td><td>New Brunswick Scientific</td><td>Innova 42</td><td></td></tr><tr><td>&amp;Auml;KTA purifier UPC 10</td><td>GE Healthcare</td><td></td><td>FPLC System</td></tr><tr><td>Fraction collector</td><td>GE Healthcare</td><td>Frac-920</td><td></td></tr><tr><td>Centrifuge (with rotor A-4-81)</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Fixed angle rotor</td><td>Eppendorf</td><td>F-34-6-38</td><td></td></tr><tr><td>Mini Protean Tetra Cell</td><td>BioRad</td><td>1658000EDU</td><td></td></tr><tr><td>NanoPhotometer</td><td>Implen</td><td>P330</td><td></td></tr><tr><td>Sonicator</td><td>Brandelin</td><td>UW2070</td><td></td></tr><tr><td>Fluorescence reader</td><td>Tecan</td><td>infinite M200PRO</td><td></td></tr><tr><td>pH meter</td><td>Knick</td><td>765</td><td></td></tr></tbody></table>

purification of human s100a12 and its ion-induced oligomers for immune cell stimulation

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.

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 TNF&#945; 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&#8217;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., &#8216;trimers&#8217;, 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 TNF&#945; release (Figure 6). Remnant cytokine release could be detected from cells stimulated with tetrameric S100A12, while treatment with dimeric protein does not induce TNF&#945; release.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60065/60065fig01.jpg" /> 
Figure 1: Results of anion-exchange chromatography.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60065/60065fig02.jpg" /> 
Figure 2: Results of hydrophobic-interaction chromatography.&#160;(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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60065/60065fig03.jpg" /> 
Figure 3: Primary human monocytes were stimulated at indicated concentrations.&#160;LPS (A) or S100A12 (wildtype, B) were left untreated or heat-denaturated (99 &#176;C, 10 min). Both conditions were tested in the presence and absence of polymyxin B. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60065/60065fig04.jpg" /> 
Figure 4: S100A12 protein was crosslinked with BS3 after incubation in HBS buffer containing 5 mM Ca2+ and indicated Zn2+ concentrations.&#160;(A) Increasing Zn2+ concentrations induce arrangement of S100A12 into tetramers and hexamers upon separation on 4&#8722;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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60065/60065fig05.jpg" /> 
Figure 5: S100A12 oligomers were separated on a size-exclusion column.&#160;(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&#8722;15% gradient SDS-PAGE. Lane 1 = dimer; lane 2 = tetramer; lane 3 = hexamer. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60065/60065fig06.jpg" /> 
Figure 6: Stimulation of monocytes with purified S100A12 oligomers.&#160;TNF&#945;-release after 4 h incubation was quantified by ELISA. The data show the mean value from two independent experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/60065fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bed Volume (CV)</td>
			<td>75 mL</td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Absorbance at 280 nM</td>
		</tr>
		<tr>
			<td>Pressure Max</td>
			<td>3 bar</td>
		</tr>
		<tr>
			<td>Column buffer A</td>
			<td>20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 8.5</td>
		</tr>
		<tr>
			<td>Column buffer B</td>
			<td>20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1 M NaCl, pH 8.5</td>
		</tr>
		<tr>
			<td>Sample Volume</td>
			<td>variable</td>
		</tr>
		<tr>
			<td>Flow Rate</td>
			<td>1&#8722;2 mL/min</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 1: Detailed information on the applied parameters of anion-exchange chromatography.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Block</td>
			<td>Volume</td>
			<td>Buffer</td>
			<td>Outlet</td>
		</tr>
		<tr>
			<td>Equilibration</td>
			<td>1&#8722;2 column volumes (CVs)</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Sample load</td>
			<td>n/a</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Wash out unbound sample</td>
			<td>1 CV</td>
			<td>A</td>
			<td>High volume outlet</td>
		</tr>
		<tr>
			<td>Gradient&#8211;Elution</td>
			<td>0&#8722;100 % Buffer B in 1 CV</td>
			<td>A to B</td>
			<td>Fraction collector</td>
		</tr>
		<tr>
			<td>Wash out&#8211;Buffer B</td>
			<td>1 CV</td>
			<td>B</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Re-Equilibration</td>
			<td>2 CVs</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
	</tbody>
</table>
Table 2: Detailed information on the used method of anion-exchange chromatography.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bed Volume (CV)</td>
			<td>125 mL</td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Absorbance at 280 nM</td>
		</tr>
		<tr>
			<td>Pressure Max</td>
			<td>4 bar</td>
		</tr>
		<tr>
			<td>Column buffer A</td>
			<td>20 mM Tris, 140 mM NaCl, 25 mM CaCl2, pH 7.5</td>
		</tr>
		<tr>
			<td>Column buffer B</td>
			<td>20 mM Tris, 140 mM NaCl, 50 mM EDTA, pH 7.5</td>
		</tr>
		<tr>
			<td>Sample Volume</td>
			<td>variable</td>
		</tr>
		<tr>
			<td>Flow Rate</td>
			<td>1&#8722;2 mL/min</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>4 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 3: Detailed information on the applied parameters of hydrophobic-interaction chromatography.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Block</td>
			<td>Volume</td>
			<td>Buffer</td>
			<td>Outlet</td>
		</tr>
		<tr>
			<td>Equilibration</td>
			<td>1&#8722;2 column volumes (CVs)</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Sample load</td>
			<td>n/a</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Wash out unbound sample</td>
			<td>1&#8722;2 CVs&#160;</td>
			<td>A</td>
			<td>High volume outlet</td>
		</tr>
		<tr>
			<td>Elution</td>
			<td>100 % Buffer B&#160;</td>
			<td>B</td>
			<td>Fraction collector</td>
		</tr>
		<tr>
			<td>Wash out&#8211;Buffer B</td>
			<td>1 CV</td>
			<td>B</td>
			<td>Waste</td>
		</tr>
		<tr>
			<td>Re-Equilibration&#160;</td>
			<td>2 CVs&#160;</td>
			<td>A</td>
			<td>Waste</td>
		</tr>
	</tbody>
</table>
Table 4: Detailed information on the used method of hydrophobic-interaction chromatography.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bed Volume (CV)</td>
			<td>320 mL</td>
		</tr>
		<tr>
			<td>Monitor</td>
			<td>Absorbance at 280 nm</td>
		</tr>
		<tr>
			<td>Pressure Max</td>
			<td>3 bar</td>
		</tr>
		<tr>
			<td>Column buffer A</td>
			<td>20 mM Hepes, 140 mM NaCl, pH 7.2</td>
		</tr>
		<tr>
			<td>Sample Volume</td>
			<td>Up to 13 mL</td>
		</tr>
		<tr>
			<td>Flow Rate</td>
			<td>1&#8722;1.5 mL/min</td>
		</tr>
		<tr>
			<td>Temperature</td>
			<td>12&#8722;15 &#176;C</td>
		</tr>
	</tbody>
</table>
Table 5: Detailed information for the applied parameters of size-exclusion chromatography.
Supplemental Table 1: Preparation of buffers and stock solutions. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60065/Supplemental_Table_Buffers_and_stock_solutions.xlsx" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation at JoVE.com

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

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

In this protocol, we describe a method to purify human calcium-binding protein S100A12 and its ion-induced oligomers from Escherichia coli culture for ...

purification-human-s100a12-its-ion-induced-oligomers-for-immune-cell

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.9K Views.  University Children's Hospital. Dit protocol beschrijft een volledige zuiveringsprocedure voor het menselijk calciumbindingeiwit, S100A12, en in het bijzonder voor het verschillende ionen-geïnduceerde oligomers voor downstream onderzoeksimplicaties. Met deze techniek zijn we in staat om een hoog rendement van zeer zuiver en endotoxinevrij eiwit te behouden. Chemische kruisverkoppeling en scheiding op de grootte uitsluiting chromatografie kolom is een overtuigende methode om homo-oligomeric eiwitten te genereren voor verder...

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

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

Biochemistry

Unraveling Key Players of Humoral Immunity: Advanced and Optimized Lymphocyte Isolation Protocol from Murine Peyer's Patches

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense against pathogens. It is well established that Peyer&#39;s patches (PPs) have an essential role in the mucosal immune network by hosting several effector T and B cell subsets. A certain fraction of these effector cells, follicular T helper (TFH) and germinal center (GC) B cells are professionalized in the regulation of humoral immunity. Hence, the characterization of these cell subsets within PPs in terms of their differentiation program and functional properties can provide important information about mucosal immunity. To this end, an easily applicable, efficient and reproducible method of lymphocyte isolation from PPs would be valuable to researchers. In this study, we aimed to generate an effective method to isolate lymphocytes from mouse PPs with high cell yield. Our approach revealed that initial tissue processing such as the use of digestive reagents and tissue agitation, as well as cell staining conditions and selection of antibody panels, have great influence on the quality and identity of the isolated lymphocytes and on experimental outcomes.
Here, we describe a protocol enabling researchers to efficiently isolate lymphocyte populations from PPs allowing reproducible flow cytometry-based assessment of T and B cell subsets primarily focusing on&#160;TFH and GC B cell subsets.

In this study, we present a novel and effective protocol for the isolation of lymphocytes from Peyer&#39;s Patches (PPs), which can be subsequently used for in vivo and in vitro functional assays as well as flow cytometric studies of follicular T helper and germinal center B cells.

In the gut mucosa, immune cells constitute a unique immunological entity, which promotes immune tolerance while concurrently conferring immune defense ...

Cancer Research

Isolating Human Peripheral Blood Mononuclear Cells and CD4+ T cells from S&#233;zary Syndrome Patients for Transcriptomic Profiling

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides (MF) and S&#233;zary syndrome (SS) represent the most common subtypes. Despite a number of studies on characterizing gene expression, genetic alterations, and epigenetic abnormalities of CTCL, the molecular pathogenesis of MF/SS remains unclear. MF refers to the more common CTCL with a skin-predominance, and is usually limited to skin, whereas&#160;SS is an aggressive leukemic variant of CTCL with widespread skin involvement and is characterized by neoplastic distribution mainly involving blood, skin, and lymph node. Focusing on clinical practice, the identification of gene expression biomarkers has&#160;enormous potential to improve diagnosis and treatment of MF/SS. Indeed, recent transcriptomic studies have identified potential diagnostic biomarkers from differences in gene expression between normal and malignant T cells, which may improve our understanding of SS biology, and reveal potential therapeutic targets. This manuscript describes a detailed reproducible protocol for the isolation of peripheral blood mononuclear cells from fresh whole blood from patients diagnosed with SS, selection of CD4+ memory T cells (CD4+CD45RO+ T cells), chemical stimulation, and preparation of RNA suitable for transcriptomic profiling to discover novel prognostic molecular markers to gain additional insight in disease etiology. The stimulation using chemical agonist to activate nuclear regulation provides more specific assessment for pathways important in the dynamic transcription regulation and gene expression and eliminates confounding defects that may arise from upstream signaling defects arising from TCR antigen loss at the cell membrane. The data obtained from comparison of transcriptome of unstimulated to stimulated SS T cells unmasks functional regulatory gene expression defects not evident from analysis of quiescent unstimulated cells. Furthermore, the method outlined from this approach can be adapted for studying T cell gene expression defects in other T cell immune diseases.

We present a simple protocol for the isolation of peripheral blood mononuclear cells from whole blood obtained from patients diagnosed with S&#233;zary Syndrome, followed by selection of CD4+ T cells, their stimulation with phorbol12-myristate13-acetate and A23187 ionophore, and preparation of RNA for transcriptomic profiling.

Cutaneous T-cell lymphomas (CTCL) are derived from the transformation and uncontrolled proliferation of mature skin-homing T cells, and mycosis fungoides ...

Purification of the Membrane Compartment for Endoplasmic Reticulum-associated Degradation of Exogenous Antigens in Cross-presentation

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I molecules also known as cross-presentation (CP). CP plays an important role not only in the stimulation of na&#239;ve CD8+ T cells and memory CD8+ T cells for infectious and tumor immunity but also in the inactivation of self-acting na&#239;ve T cells by T cell anergy or T cell deletion. Although the critical molecular mechanism of CP remains to be elucidated, accumulating evidence indicates that exogenous antigens are processed through endoplasmic reticulum-associated degradation (ERAD) after export from non-classical endocytic compartments. Until recently, characterizations of these endocytic compartments were limited because there were no specific molecular markers other than exogenous antigens. The method described here is a new vesicle isolation protocol, which allows for the purification of these endocytic compartments. Using this purified microsome, we reconstituted the ERAD-like transport, ubiquitination, and processing of the exogenous antigen in vitro, suggesting that the ubiquitin-proteasome system processed the exogenous antigen after export from this cellular compartment. This protocol can be further applied to other cell types to clarify the molecular mechanism of CP.

The method described here is a new vesicle isolation protocol, which allows for the purification of the cellular compartments where exogenous antigens are processed by endoplasmic reticulum-associated degradation in cross-presentation.

Dendritic cells (DCs) are highly capable of processing and presenting internalized exogenous antigens upon major histocompatibility class (MHC) I ...

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different infectious diseases. In this article we demonstrate a method to produce a SAPN core containing a six-helix bundle (SHB) assembly that is capable of presenting antigens in a trimeric conformation. We describe the expression of the SHB-SAPN in an E. coli system, as well as the necessary protein purification steps. We included an isopropanol wash step to reduce the residual bacterial lipopolysaccharide. As an indication of the protein identity and purity, the protein reacted with known monoclonal antibodies in Western blot analyses. After refolding, the size of the particles fell in the expected range (20 to 100 nm), which was confirmed by dynamic light scattering, nanoparticle tracking analysis, and transmission electron microscopy. The methodology described here is optimized for the SHB-SAPN, however, with only slight modifications it can be applied to other SAPN constructs. This method is also easily transferable to large scale production for GMP manufacturing for human vaccines.

Production of E. coli-expressed Self-Assembling Protein Nanoparticles for Vaccines Requiring Trimeric Epitope Presentation

A detailed method is provided here describing the purification, refolding, and characterization of self-assembling protein nanoparticles (SAPNs) for use in vaccine development.

Self-assembling protein nanoparticles (SAPNs) function as repetitive antigen displays and can be used to develop a wide range of vaccines for different ...

Bioengineering

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

Biology

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Myeloid Innate Signaling Pathway Regulation by MALT1 Paracaspase Activity

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells downstream of pattern recognition receptors. Best studied are the Dectin-1 and Dectin-2 members of the C-type lectin-like receptor family that induce a SYK- and CARD9-dependent signaling cascade leading to NF-&#954;B activation, in a MALT1-dependent manner. By contrast, Toll-like receptors (TLR), such as TLR-4, propagate NF-&#954;B activation but signal via an MYD88/IRAK-dependent cascade. Nonetheless, whether MALT1 might contribute to TLR-4 signaling has remained unclear. Recent evidence with MLT-827, a potent and selective inhibitor of MALT1 paracaspase activity, indicates that TNF- production downstream of TLR-4 in human myeloid cells is independent of MALT1, as opposed to TNF- production downstream of Dectin-1, which is MALT1 dependent. Here, we addressed the selective involvement of MALT1 in pattern recognition sensing further, using a variety of human and mouse cellular preparations, and stimulation of Dectin-1, MINCLE or TLR-4 pathways. We also provided additional insights by exploring cytokines beyond TNF-, and by comparing MLT-827 to a SYK inhibitor (Cpd11) and to an IKK inhibitor (AFN700). Collectively, the data provided further evidence for the MALT1-dependency of C-type lectin-like receptor &#8212;signaling by contrast to TLR-signaling.

MALT1 regulates innate immunity but how this occurs remains ill-defined. We used the selective MALT1 paracaspase inhibitor MLT-827 to unravel the contribution of MALT1 to innate signaling downstream of Toll-like or C-type lectin-like receptors, demonstrating that MALT1 regulates the production of myeloid cytokines, and downstream of C-type lectin-like receptors, selectively.

Besides its function in lymphoid cells, which has been addressed by numerous studies, the paracaspase MALT1 also plays an important role in innate cells ...

Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Source: Fuehner, S., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60065">Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation</a> J. Vis. Exp. (2019)
In this video, we demonstrate an anion-exchange chromatography technique to purify human calcium-binding protein S100A12 based on the net surface charge of the protein from an Escherichia coli culture.

Source: Fuehner, S., et al. Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation J. Vis. Exp. (2019)
In this video, we ...

JoVE Encyclopedia of Experiments

Biological Techniques

Chromatography Techniques

Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

Source: Fuehner, S., et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/60065">Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation</a>. J. Vis. Exp. (2019).
In this video, we demonstrate the purification of calcium-binding protein from a dialyzed cell lysate through calcium-dependent hydrophobic interaction chromatography. The calcium-binding proteins expose a hydrophobic region upon binding with calcium, facilitating interaction with a hydrophobic group on resin. Later these proteins are eluted using calcium chelator EDTA that reverses the interaction.

Source: Fuehner, S., et al., Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation. J. Vis. Exp. (2019).
In this video, ...

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

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