We would like to thank Dr. Tom Kirby, Scott Gabel, and Dr. Robert London for their help and assistance throughout this work, along with Dr. Bob Petrovich and Lori Edwards for the use of their instrumentation and their assistance in generating the Bla g 1 constructs employed in this study. We thank Andrea Adams for assistance with the mass spectrometry, and Dr. Eugene DeRose for assistance with the NMR instrumentation. This research was supported by the Intramural Research Program of the &#65279;NIH, National Institute of Environmental Health Sciences, Z01-ES102906 (GAM). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National &#65279;Institute of Environmental Health Sciences.

1. Bla g 1 cloning
<ol>
	<li>Obtain gene for cockroach allergen Bla g 1.0101 (residues 34-216), representing a single repeat of the MA domain. For the sake of simplicity, Bla g 1 will be used throughout the work to represent this single repeat, rather than the entire Bla g 1.0101 transcript.</li>
	<li>Subclone the Bla g 1 gene into the desired vector. In this study, the gene containing an N-terminal glutathione S-transferase (GST) tag coupled to a tobacco etch virus (TEV) protease cleavage site was inserted into a pGEX vector for expression as described previously11.</li>
	<li>Transform the Bla g 1 pGEX vector into BL21 DE3 E. coli cells.
	<ol>
		<li>Prepare a 10 ng/&#181;L stock of the desired vector.</li>
		<li>Combine 1 &#181;L of 10 ng/&#181;L DNA stock with 50 &#181;L of BL21 DE3 cells as provided by the manufacturer.</li>
		<li>Incubate BL21 DE3-DNA mixture for 30 min on ice. Transfer to a 42 &#730;C water bath for 1 min, then immediately transfer back on ice for an additional 1 min incubation.</li>
		<li>Add 200 &#181;L of LB media to the cells and incubate for an additional 1 h at 37 &#730;C.</li>
		<li>Plate the transformed cells on LB-Agar plates containing 100 mg/L ampicillin and grow at 37 &#730;C overnight.</li>
	</ol>
	</li>
</ol>
2. Initial expression and purification
<ol>
	<li>Inoculate 1 L of LB media containing 100 mg/L ampicillin with a single colony of BL21 DE3 cells transformed with the Bla g 1 vector as described in 1.3. Grow at 37 &#730;C overnight.</li>
	<li>On the next day, harvest cells (OD600 ~1.5) via centrifugation at 6,000 x g for 10 min and resuspend in 2 L of 2x YT media containing 100 mg/L ampicillin. Allow cells to grow for an additional 1 h at 37 &#730;C to an OD600 &#62; 0.6.</li>
	<li>Induce protein expression through the addition of 0.5 mM IPTG. Transfer cells to 18 &#730;C and incubate overnight.</li>
	<li>On the next day, harvest cells as described in 2.2. The resulting cell pellet can be frozen and stored at -20 &#730;C.</li>
	<li>Resuspend pellet obtained from 1 L of culture in 50 mL of lysis buffer (50 mM Tris-HCl pH 8.5, 100 mM NaCl) containing 1 protease inhibitor tablet (or equivalent) and 1 &#181;L of benzonase nuclease.</li>
	<li>Lyse cells using a probe sonicator (500 W, 20 kHz) set to 30&#8211;50% power for 4 min with a 50% duty cycle. Keep the lysate in an ice bath during sonication</li>
	<li>Centrifuge lysate at 45,000 x g for 20 min. Discard insoluble fraction (pellet).
	<ol>
		<li>Remove 28 &#181;L of soluble protein. Combine with 7 &#181;L of 5x SDS-PAGE buffer and store for SDS-PAGE analysis. Repeat this step for the GST column flow-through, wash, and elution fractions before and after incubation with TEV.</li>
	</ol>
	</li>
	<li>Apply soluble proteins (supernatant) to a glutathione resin column (~10 mL total bed volume) equilibrated in PBS pH 7.4.</li>
	<li>Wash out any unbound proteins using 50 mL of PBS.</li>
	<li>Elute GST-Bla g 1 using 50 mL of PBS containing 10 mM reduced glutathione.</li>
	<li>Incubate eluted protein with 0.2 kU TEV protease overnight at 4 &#730;C, or room temperature for 6 h to remove GST tag.</li>
</ol>
3. Endogenous lipid removal via reverse-phase HPLC
<ol>
	<li>Collect the cleaved Bla g 1 and concentrate it to ~2 mL using a centrifugal filter unit with a &#60;10 kDa molecular weight cut-off.
	<ol>
		<li>Add &#60;12 mL sample to the top of concentrator and spin at 5,000 x g for 10&#8211;15 min in a swing-bucket rotor. 
		NOTE: Sample volume and spin speed will vary based on the specific filter and the type of rotor employed. Consult manufacturer documentation prior to use.</li>
	</ol>
	</li>
	<li>Load the concentrate onto a 250 x 10 mm HPLC system equipped with a C18 reverse-phase chromatography column equilibrated with 97% buffer A (water, 0.1% trifluoroacetic acid) and 3% buffer B (acetonitrile, 0.1% trifluoroacetic acid). 
	NOTE: Smaller columns may be used, but protein may have to be loaded and eluted using multiple cycles to accommodate the reduced binding capacity. When selecting a column ensure that the resin beads have a particle size of &#60; 5 &#956;m and pore size of &#62;200 &#197; to permit effective separation of protein-sized molecules 
	CAUTION: Trifluoroacetic acid is highly corrosive and should be dispensed within a fume hood using appropriate PPE (i.e., nitrile gloves, lab coat and goggles). Acetonitrile is both moderately toxic, volatile, and highly flammable, should be used and dispensed within a fume hood using appropriate PPE (i.e., nitrile gloves, lab coat and goggles).</li>
	<li>Elute Bla g 1 using the protocol shown in Table 1 at a flow rate of 1.5&#8211;4.0 mL/min. Monitor the elution process using the fluorescence absorbance at 280 nm.
	<ol>
		<li>Collect and pool Bla g 1 fractions. Bla g 1 normally elutes at &#62;74% buffer B, or ~34&#8211;40 min. 
		NOTE: Elution time will vary slightly depending on the flow rate or column size. Collect fractions based on A280 for best results.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Time (Min)</td>
			<td>Buffer A (%)</td>
			<td>Buffer B (%)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>97</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>97</td>
			<td>3</td>
		</tr>
		<tr>
			<td>25</td>
			<td>35</td>
			<td>65</td>
		</tr>
		<tr>
			<td>55</td>
			<td>5</td>
			<td>95</td>
		</tr>
		<tr>
			<td>65</td>
			<td>5</td>
			<td>95</td>
		</tr>
		<tr>
			<td>70</td>
			<td>97</td>
			<td>3</td>
		</tr>
	</tbody>
</table>
Table 1: Elution protocol for Bla g 1. Table illustrating the elution gradient employed in the isolation of Bla g 1 using a C18 HPLC column.
<ol start="4">
	<li>Aliquot the sample into glass test tubes, filling no test tube more than halfway (~4 mL). Cover tubes with paraffin film and perforate the covering with two holes to allow venting.
	<ol>
		<li>Prepare a separate 1 mL aliquot (test aliquot). This will be used to determine the expected yield.</li>
	</ol>
	</li>
	<li>Freeze the samples and test aliquot by placing them in a -80 &#730;C freezer for 1 h, or immersion in liquid nitrogen. In the case of the later, the tube must be rotated continuously to avoid test tube breakage due to expansion of the liquid phase upon freezing.</li>
	<li>Dry the resulting delipidated protein samples using a lyophilizer. Dried protein may be stored at 4 &#730;C for several months in a sealed container.</li>
</ol>
4. Reconstitution of Apo- and cargo-loaded Bla g 1
<ol>
	<li>Determine the anticipated Bla g 1 yield.
	<ol>
		<li>Resuspend lyophilized, delipidated (post-HPLC) test aliquot in 5 mL of refolding buffer, (50 mM HEPES pH 7.4, 100 mM NaCl, 2% DMSO).</li>
		<li>Heat the mixture in a water bath (500 mL beaker with 250 mL water and stir bar over a hot plate) to 95 &#730;C. Vortex solutions intermittently and incubate at 95 &#730;C for 0.5&#8211;1 h.</li>
		<li>Remove heat and slowly let the water bath equilibrate to room temperature (~1 h). Annealed protein can be stored in this form overnight at 4 &#730;C if needed.</li>
		<li>Pass annealed Bla g 1-lipid mixture through a 0.22 &#181;M syringe filter to remove particulate matter.</li>
		<li>Buffer exchange the filtered protein 3x into PBS pH 7.4 using a centrifugal filter with 10 kDa cutoff as discussed in 3.1 to remove residual free fatty acids and organic solvent.</li>
		<li>Assess protein concentration using BCA assay or other preferred method such as UV absorbance. Use this to determine the anticipated yield for the remaining Bla g 1 aliquots.</li>
	</ol>
	</li>
	<li>Reconstitute Apo-&#160; or cargo-loaded Bla g 1
	<ol>
		<li>Resuspend Bla g 1 aliquots in refolding buffer as described in 4.1.1.</li>
		<li>To produce Apo-Bla g 1, repeat steps 4.1.2&#8211;4.1.6 to obtain desired yield.</li>
		<li>To load Bla g 1 with fatty acids, prepare 20 mM stock solutions of the desired fatty acid cargo in methanol or DMSO.&#160; Then, skip to step 4.2.5.</li>
		<li>To load Bla g 1 with phospholipids, prepare a 10 mg/mL stock of the desired cargo in chloroform inside a glass test tube.
		<ol>
			<li>Evaporate the chloroform to produce a lipid film. Add PBS to the test tube to produce a final phospholipid concentration of 20 mM. 
			CAUTION: Chloroform is harmful if inhaled or swallowed. Use in a chemical fume hood or employ respirator if inadequate ventilation is available. Employ nitrile gloves, lab coat and goggles when handling. Consult MSDS prior to use.</li>
			<li>Rehydrate the lipid film by heating it above the phase transition temperature of the lipid cargo and vortexing until the solution turns cloudy. Note that sonication may be required to fully resuspend and rehydrate some cargoes.</li>
			<li>If sonication is required, place the test tube in bath sonicator (100 W, 42 kHz) and sonicate at maximum power until cargo is resuspended. Alternatively, a probe sonicator (described in 2.6) may be used an 10&#8211;20% power with a 50% duty cycle. 
			CAUTION: Sonication employs high frequency sound waves which may damage hearing. Employ noise-suppressing PPE (earplugs or mufflers). If possible, place sonicator inside sound-dampening cabinet or chamber.</li>
		</ol>
		</li>
		<li>Add the desired fatty acid or phospholipid cargo to produce a 20x molar excess of ligands relative to Bla g 1 based on the anticipated yield determined in 4.1. The total volume of organic solvent added in this step should not exceed 2%. Vortex to mix. 
		NOTE: 1 L of Bl 21 DE3 cells typically yields ~0.25&#8211;0.4 nmol protein, corresponding to ~400 &#181;M ligand per tube.</li>
		<li>Anneal the protein as described in 4.1.</li>
	</ol>
	</li>
</ol>
5. Confirming phospholipid cargo removal/loading via 31P-NMR
<ol>
	<li>Concentrate samples of Apo- or cargo-loaded Bla g 1 to &#62;100 &#181;M using a centrifugal filter unit as described in 3.1.</li>
	<li>Rehydrate reference phospholipid in PBS buffer to final concentrations of 2, 1.5, 1, 0.5, and 0.25 mM.</li>
	<li>Dilute samples 1:1 with cholate buffer (100 mM Tris pH 8.0, 100 mM NaCl, 10% w/v cholate) to a total volume of ~600 &#181;L. 
	NOTE: Cholate is employed in this step to fully extract and solubilize lipids from the Bla g 1 hydrophobic cavity. This ensures that the chemical environment surrounding the phospholipid headgroups is consistent between different samples, allowing for its quantitative assessment using 31P-NMR. The use of cholate can be substituted for &#160;chloroform/methanol as described previously15.</li>
	<li>Acquire 1D 31P-NMR spectra of the cholate-solubilized Bla g 1 samples and reference phospholipid standards using a broadband probe. 
	NOTE: The 31P-NMR spectra presented in this work were obtained using a 600 MHz spectrometer. However, previous studies employing similar techniques suggests that acceptable sensitivity can be achieved at fields strengths as low as 150-200 MHz15.</li>
	<li>Process the resulting data using appropriate software16.</li>
	<li>Obtain peak intensities using preferred NMR viewing software17.</li>
	<li>Compare the Bla g 1 31P-NMR spectra to those obtained for the phospholipid reference samples to confirm removal of endogenously bound ligands and/or binding of desired ligands based on the chemical shifts of the visible peaks (or lack thereof).
	<ol>
		<li>Confirm full binding stoichiometry by comparing the peak intensity of the Bla g 1 spectrum to that of the phospholipid reference standards.</li>
	</ol>
	</li>
</ol>
6. Confirming Bla g 1 folding
<ol>
	<li>Prepare 0.5 &#181;M samples of Bla g 1 in CD buffer (100 mM KH2PO4, buffer pH 7.5). Load 2&#160;mL of the sample into a 10 mm CD cuvette with magnetic stir bar.</li>
	<li>Measure CD spectrum of Bla g 1 to confirm reconstitution of secondary structure. Ensure that Photomultiplier (PMT) voltage&#160;does not exceed manufacturer recommendations (generally 1 kV).
	<ol>
		<li>Measure CD signal from 260&#8211;200 nm at 25 &#730;C with a data pitch of 0.2 nm and a scan rate of 20 nm/s with a data integration time of 1 s.</li>
	</ol>
	</li>
	<li>Increase the temperature in the CD cell from 25 &#730;C&#8211;95 &#730;C at a rate of 0.5 &#730;C/min. Activate magnetic stir bar to ensure temperature is uniform across the sample.</li>
	<li>Monitor CD at 222 nm, taking readings every 2 &#730;C.</li>
	<li>Fit resulting data to a 2-state Boltzman curve to determine the melting temperature. Due to the high stability of Bla g 1, the melting temperature (MT25) was defined as the temperature at which the protein has lost 25% of its initial CD at 222 nm.</li>
</ol>

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N., 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=Are+dust+mite+allergens+more+abundant+and/or+more+stable+than+other+Dermatophagoides+pteronyssinus+proteins.">Are dust mite allergens more abundant and/or more stable than other Dermatophagoides pteronyssinus proteins.</a> Journal of Allergy and Clinical Immunology. 139 (3), 1030-1032 (2017).</li><li>Cabrera, A., 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=Are+allergens+more+abundant+and/or+more+stable+than+other+proteins+in+pollens+and+dust.">Are allergens more abundant and/or more stable than other proteins in pollens and dust.</a> Allergy: European Journal of Allergy and Clinical Immunology. , 1267-1269 (2019).</li><li>Offermann, L. R., 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=Structural+and+functional+characterization+of+the+hazelnut+allergen+Cor+a+8.">Structural and functional characterization of the hazelnut allergen Cor a 8.</a> Journal of Agricultural and Food Chemistry. 63 (41), 9150-9158 (2015).</li><li>Koppelman, S. J., 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=Reversible+denaturation+of+Brazil+nut+2S+albumin+(Ber+e1)+and+implication+of+structural+destabilization+on+digestion+by+pepsin.">Reversible denaturation of Brazil nut 2S albumin (Ber e1) and implication of structural destabilization on digestion by pepsin.</a> Journal of Agricultural and Food Chemistry. 53 (1), 123-131 (2005).</li><li>Smole, U., Bublin, M., Radauer, C., Ebner, C., Breiteneder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mal+d+2,+the+thaumatin-like+allergen+from+apple,+is+highly+resistant+to+gastrointestinal+digestion+and+thermal+processing.">Mal d 2, the thaumatin-like allergen from apple, is highly resistant to gastrointestinal digestion and thermal processing.</a> International Archives of Allergy and Immunology. 147 (4), 289-298 (2008).</li><li>Bublin, M., 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=Effects+of+gastrointestinal+digestion+and+heating+on+the+allergenicity+of+the+kiwi+allergens+Act+d+1,+actinidin,+and+Act+d+2,+a+thaumatin-like+protein.">Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein.</a> Molecular Nutrition and Food Research. 52 (10), 1130-1139 (2008).</li><li>Griesmeier, U., 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=Physicochemical+properties+and+thermal+stability+of+Lep+w+1,+the+major+allergen+of+whiff.">Physicochemical properties and thermal stability of Lep w 1, the major allergen of whiff.</a> Molecular Nutrition and Food Research. 54 (6), 861-869 (2010).</li><li>de Jongh, H. H. J., 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=Effect+of+heat+treatment+on+the+conformational+stability+of+intact+and+cleaved+forms+of+the+peanut+allergen+Ara+h+6+in+relation+to+its+IgE-binding+potency.">Effect of heat treatment on the conformational stability of intact and cleaved forms of the peanut allergen Ara h 6 in relation to its IgE-binding potency.</a> Food Chemistry. 326, 127027 (2020).</li><li>Glasgow, B. J., Abduragimov, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+complexes+in+lipocalins:+Underestimation+of+the+stoichiometry+parameter+(n).">Ligand binding complexes in lipocalins: Underestimation of the stoichiometry parameter (n).</a> Biochimica et Biophysica Acta - Proteins and Proteomics. 1866 (10), 1001-1007 (2018).</li><li>Aalberse, R. C., 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=Identification+of+the+amino-terminal+fragment+of+Ara+h+1+as+a+major+target+of+the+IgE-binding+activity+in+the+basic+peanut+protein+fraction.">Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.</a> Clinical and Experimental Allergy. 50 (3), 401-405 (2020).</li><li>Bublin, M., Eiwegger, T., Breiteneder, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+lipids+influence+the+allergic+sensitization+process.">Do lipids influence the allergic sensitization process.</a> Journal of Allergy and Clinical Immunology. 134 (3), 521-529 (2014).</li></ol>

The authors have nothing to disclose.

The protocol described in this work has been successfully applied to systematically study the lipid binding properties of Bla g 1. This revealed a correlation between cargo binding, thermostability, and endosomal processing, the latter of which was correlated with decrease in the generation of a known T-cell epitope with potential implications for immunogenicity9,18. In addition to Bla g 1, other allergens such as Pru p 3 and Bet v 1 have been shown to retain the...

A survey of the allergen database reveals that allergens are found in only 2% of all known protein families, suggesting common functional and biophysical properties contribute to allergenicity1. Of these properties, the ability to bind lipid cargoes appears to be strongly over-represented among allergens, suggesting that these cargoes may influence the sensitization process1. Indeed, it has been shown that the Brazil Nut allergen Ber e 1 requires co-administration with its endogenous lipid to realize its full sensitizing potential2. These lipids could potentially stimulate the immune system directly as illustrated by the mite allergens Der p 2 and Der p 7, both of which share a strong structural homology with LPS-binding proteins3,4,5. Based on this observation it was proposed that Derp 2 and Der p 7 could bind bacterial lipids and directly stimulate the host immune system through TLR4-mediated signaling, facilitating the sensitization process5,6. It is also possible that endogenously bound lipids could alter the biophysical properties of allergenic&#160;proteins themselves. For example, the ability of Sin a 2 (mustard) and Ara h 1 (peanuts) to interact with phospholipid vesicles significantly enhanced their resistance to gastric and endosomal degradation7, while ligand binding to the major birch pollen allergen Bet v 1 altered both the rate of endosomal processing and the diversity of the resulting peptides8. This is particularly relevant to allergenicity given the correlation that has been observed between stability, T-cell epitope generation and allergenicity for proteins such as Bet v 1 and Bla g 1; the latter of which will be the subject of this work9,10.
Bla g 1 represents the prototypical member of the insect Major Allergen (MA) protein family, and possesses a unique structure composed of 12 amphipathic alpha helices which enclose an abnormally large hydrophobic cavity9,11. The available X-ray crystal structure of Bla g 1 shows electron density within this cavity consistent with bound phospholipid or fatty acid ligands; a conjecture confirmed by 31P-NMR and mass spectrometry. These cargoes were heterogeneous in nature and their composition was heavily dependent on the allergen source, with different lipid profiles observed for recombinant Bla g 1 expressed in E. coli and P. pastoris. Curiously, Bla g 1 purified from its natural allergen source (cockroach frass) contained predominantly fatty acids within its binding site, with a mixture of palmitate, oleate, and stearate being identified as its &#8220;natural&#8221; ligands9,11. The ability of Bla g 1 to retain lipids and fatty acids following multiple purification steps hinders efforts to study the protein in isolation. Conversely, it has been suggested that the natural palmitate, stearate, and oleate ligands of Bla g 1 (henceforth referred to as nMix) play a key role in both its allergenicity and native biological function9. However, these ligands are not present in Bla g 1 obtained from recombinant sources, making it difficult to assess this hypothesis. Similar issues have been observed for other lipid binding allergens such as Bet v 112,13. To facilitate the systematic study of lipid-allergen interactions we have developed a protocol through which allergens can be quantitatively stripped of their endogenously bound lipids and reconstituted in either Apo-form or loaded with specific ligands.
Allergens are most commonly purified from their natural or recombinant sources using affinity chromatography and/or size-exclusion chromatography. Here, we introduce an additional purification step in the form of high-performance liquid chromatography (HPLC) employing a reverse-phase C18 column from which the allergen is eluted into an organic solvent similar to protocols developed for fatty acid binding proteins14. The resulting protein is then subjected to a thermal annealing step in the absence or presence of fatty acids and/or phospholipids. In addition to recovering the native Bla g 1 fold, the elevated temperatures increase the solubility and accessibility of the lipid cargoes, yielding Bla g 1 in either the Apo-form or uniformly loaded with the desired hydrophobic ligand. 31P-NMR spectra of Bla g 1 purified in this manner confirmed&#160;the complete removal of endogenously bound ligands and uniform replacement with the desired compounds, while circular dichroism confirmed the successful recovery of the Bla g 1 fold. The utility of this method is highlighted in a recent work in which cargo binding was found to enhance Bla g 1 thermostability and proteolytic resistance, altering the kinetics of T-cell epitope generation with potential implications for sensitization and allergenicity9.

<table><tbody><tr><td>Bla g 1 Gene&amp;nbsp;</td><td>Genescript</td><td>N/a</td><td>Custom gene synthesis service. GenBank Accession no AF072219 Residues 34-216</td></tr><tr><td>Affinity purified natural Bla g 1 (nBla g 1)</td><td>Indoor biotechnologies</td><td>N/a</td><td>Custom order</td></tr><tr><td>Agilent 1100 Series HPLC System</td><td>Agilent</td><td>G1315B, G1311A, G1322A</td><td>UV Detector, Pump, and Degasser</td></tr><tr><td>Agilent DD2 600 MHz spectrometer</td><td>Agilent</td><td>N/a</td><td></td></tr><tr><td>Amicon Ultra-15 Centrifugal Filter Unit</td><td>Amicon</td><td>UFC-1008</td><td></td></tr><tr><td>Ampicillin</td><td>Fisher Scientific</td><td>BP1760-5</td><td></td></tr><tr><td>Benzonase</td><td>Sigma-Aldrich</td><td>E1014-5KU</td><td></td></tr><tr><td>Broad- band 5 mm Z-gradient probe</td><td>Varian</td><td>N/a</td><td></td></tr><tr><td>ChemStation for LC (Software)</td><td>Agilent</td><td>N/a</td><td></td></tr><tr><td>cOmplete Mini Protease Inhibitor Cocktail</td><td>Roche</td><td>11836153001</td><td></td></tr><tr><td>Distearoylphosphatidylcholine (18:0 PC)</td><td>Avanti Polar Lipids</td><td>850365C</td><td></td></tr><tr><td>E. Coli BL21 DE3 Cells</td><td>New England Biolabs</td><td>C2530H</td><td></td></tr><tr><td>Freezone 4.5 Freeze Dry System</td><td>Labconco</td><td>7750000</td><td></td></tr><tr><td>Glutathione Resin</td><td>Genescript</td><td>L00206</td><td></td></tr><tr><td>Glutathione, Reduced</td><td>Fisher Scientific</td><td>BP25211</td><td></td></tr><tr><td>Isopropyl-&amp;beta;-D-thiogalactopyranoside (IPTG)</td><td>Fisher Scientific</td><td>34060</td><td></td></tr><tr><td>Jasco&amp;nbsp; CD spectropolarimeter</td><td>Jasco</td><td>J-815</td><td></td></tr><tr><td>Millex Syringe Filter Unit</td><td>EMD Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td>NMRPipe (Software)</td><td>Delaglio et al.&amp;nbsp;</td><td>N/a</td><td>Delaglio, F. et al. Nmrpipe - a Multidimensional Spectral Processing System Based On Unix Pipes. J. Biomol. NMR 6, 277&amp;ndash;293 (1995).</td></tr><tr><td>NMRViewJ (Software)</td><td>Johnson et al.&amp;nbsp;</td><td>N/a</td><td>Johnson, B. A. &amp;amp; Blevins, R. A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603&amp;ndash;614 (1994).</td></tr><tr><td>Oleic acid</td><td>Sigma-Aldrich</td><td>O1008</td><td></td></tr><tr><td>Pierce BCA Protein Assay</td><td>Sigma-Aldrich</td><td>BCA1-1KT</td><td></td></tr><tr><td>Polaris 5 C18-A 250x10.0 mm HPLC Column</td><td>Agilent</td><td>SKU: A2000250X100</td><td></td></tr><tr><td>SD-200 Vacuum Pump</td><td>Varian</td><td>VP-195</td><td></td></tr><tr><td>Sodium Cholate Hydrate</td><td>Sigma-Aldrich</td><td>C6445</td><td></td></tr><tr><td>Sodium Palmitate</td><td>Sigma-Aldrich</td><td>P9767</td><td></td></tr><tr><td>Sodium Stearate</td><td>Sigma-Aldrich</td><td>S3381</td><td></td></tr><tr><td>VnmrJ (Software)</td><td>Varian</td><td>N/a</td><td></td></tr></tbody></table>

removal and replacement of endogenous ligands from lipid-bound proteins and allergens

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and have the potential to influence the sensitization process either through directly stimulating the immune system or altering the biophysical properties of the allergenic protein. In order to control for these variables, techniques are required for the removal of endogenously bound ligands and, if necessary, replacement with lipids of known composition. The cockroach allergen Bla g 1 encloses a large hydrophobic cavity which binds a heterogeneous mixture of endogenous lipids when purified using traditional techniques. Here, we describe a method through which these lipids are removed using reverse-phase HPLC followed by thermal annealing to yield Bla g 1 in either its Apo-form or reloaded with a user-defined mixture of fatty acid or phospholipid cargoes. Coupling this protocol with biochemical assays reveal that fatty acid cargoes significantly alter the thermostability and proteolytic resistance of Bla g 1, with downstream implications for the rate of T-cell epitope generation and allergenicity. These results highlight the importance of lipid removal/reloading protocols such as the one described herein when studying allergens from both recombinant and natural sources. The protocol is generalizable to other allergen families including lipocalins (Mus m 1), PR-10 (Bet v 1), MD-2 (Der p 2) and Uteroglobin (Fel d 1), providing a valuable tool to study the role of lipids in the allergic response.

Using affinity chromatography, recombinant GST-Bla g 1 was readily isolated to a high level of purity (Figure 1A), producing a yield of ~2&#8211;4 mg/L of cell culture. Overnight incubation with TEV protease at 4 &#730;C is sufficient to remove the GST tag, yielding the final product at ~24 kDa. Note that in this instance there is a significant amount of GST-Bla g 1 in the flow-through and wash fractions, suggesting the Glutathione resin binding capacity was exceeded. The use of more resin or multiple cy...

Watch this Scientific Journal Video about Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens at JoVE.com

Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Many major allergens bind to hydrophobic lipid-like molecules, including Mus m 1, Bet v 1, Der p 2, and Fel d 1. These ligands are strongly retained and ...

removal-replacement-endogenous-ligands-from-lipid-bound-proteins

Research

JoVE Journal

Biochemistry

2.9K Views.  National Institute of Environmental Health Sciences. This protocol describes the removal of endogenous lipids from allergens, and their replacement with user-specified ligands through reverse-phase HPLC coupled with thermal annealing. 31P-NMR and circular dichroism allow for the rapid confirmation of ligand removal/loading, and the recovery of native allergen structure.

Video: Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.

Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses.

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

Chemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Ligand Nano-cluster Arrays in a Supported Lipid Bilayer

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological studies. In the past years, it has become increasingly clear that living cells respond, not only to the biochemical nature of the molecules presented to them but also to the way these molecules are presented. Creating protein micro-patterns is therefore now standard in many biology laboratories; nano-patterns are also more accessible. However, in the context of cell-cell interactions, there is a need to pattern not only proteins but also lipid bilayers. Such dual proteo-lipidic patterning has so far not been easily accessible. We offer a facile technique to create protein nano-dots supported on glass and propose a method to backfill the inter-dot space with a supported lipid bilayer (SLB). From photo-bleaching of tracer fluorescent lipids included in the SLB, we demonstrate that the bilayer exhibits considerable in-plane fluidity. Functionalizing the protein dots with fluorescent groups allows us to image them and to show that they are ordered in a regular hexagonal lattice. The typical dot size is about 800 nm and the spacing demonstrated here is 2 microns. These substrates are expected to serve as useful platforms for cell adhesion, migration and mechano-sensing studies.

We present a protocol to functionalize glass with nanometric protein patches surrounded by a fluid lipid bilayer. These substrates are compatible with advanced optical microscopy and are expected to serve as platform for cell adhesion and migration studies.

Currently there is considerable interest in creating ordered arrays of adhesive protein islands in a sea of passivated surface for cell biological ...

Bioengineering

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 ...

Immunology and Infection

Enrichment of Bacterial Lipoproteins and Preparation of N-terminal Lipopeptides for Structural Determination by Mass Spectrometry

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their significance to both cell physiology and immunology, much remains to be discovered about novel lipoprotein forms, how they are synthesized, and the effect of the various forms on host immunity. To enable thorough studies on lipoproteins, this protocol describes a method for bacterial lipoprotein enrichment and preparation of N-terminal tryptic lipopeptides for structural determination by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Expanding on an established Triton X-114 phase partitioning method for lipoprotein extraction and enrichment from the bacterial cell membrane, the protocol includes additional steps to remove non-lipoprotein contaminants, increasing lipoprotein yield and purity. Since lipoproteins are commonly used in Toll-like receptor (TLR) assays, it is critical to first characterize the N-terminal structure by MALDI-TOF MS. Herein, a method is presented to isolate concentrated hydrophobic peptides enriched in N-terminal lipopeptides suitable for direct analysis by MALDI-TOF MS/MS. Lipoproteins that have been separated by Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis (SDS-PAGE) are transferred to a nitrocellulose membrane, digested in situ with trypsin, sequentially washed to remove polar tryptic peptides, and finally eluted with chloroform-methanol. When coupled with MS of the more polar trypsinized peptides from wash solutions, this method provides the ability to both identify the lipoprotein and characterize its N-terminus in a single experiment. Intentional sodium adduct formation can also be employed as a tool to promote more structurally informative fragmentation spectra. Ultimately, enrichment of lipoproteins and determination of their N-terminal structures will permit more extensive studies on this ubiquitous class of bacterial proteins.

The enrichment of bacterial lipoproteins using a non-ionic surfactant phase partitioning method is described for direct use in TLR assays or other applications. Further steps are detailed to prepare N-terminal tryptic lipopeptides for structural characterization by mass spectrometry.

Lipoproteins are important constituents of the bacterial cell envelope and potent activators of the mammalian innate immune response. Despite their ...

Antigenic Liposomes for Generation of Disease-specific Antibodies

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for vaccination as well as understand how pathogenic antibody responses develop in allergies and autoimmune disease. Generating robust antigen-specific antibody responses is not always trivial. In mouse models, it often requires multiple rounds of immunizations with adjuvant that leads to a great deal of variability in the levels of induced antibodies. One example is in mouse models of peanut allergies where more robust and reproducible models that minimize mouse numbers and the use of adjuvant would be beneficial. Presented here is a highly reproducible mouse model of peanut allergy anaphylaxis. This new model relies on two key factors: (1) antigen-specific splenocytes are adoptively transferred from a peanut-sensitized mouse into a na&#239;ve recipient mouse, normalizing the number of antigen-specific memory B- and T-cells across a large number of mice; and (2) recipient mice are subsequently boosted with a strong multivalent immunogen in the form of liposomal nanoparticles displaying the major peanut allergen (Ara h 2). The major advantage of this model is its reproducibility, which ultimately lowers the number of animals used in each study, while minimizing the number of animals receiving multiple injections of adjuvant. The modular assembly of these immunogenic liposomes provides relatively facile adaptability to other allergic or autoimmune models that involve pathogenic antibodies.

Described is the preparation of antigenic liposomal nanoparticles and their use in stimulating B-cell activation in vitro and in vivo. Consistent and robust antibody responses led to the development of a new peanut allergy model. The protocol for generating antigenic liposomes can be extended to different antigens and immunization models.

Antibody responses provide critical protective immunity to a wide array of pathogens. There remains a high interest in generating robust antibodies for ...

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.

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 ...

Humanized Mediator Release Assay as a Read-Out for Allergen Potency

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells and basophils, upon stimulation with serial dilutions of putative allergens. Therefore, these assays represent an essential tool that mimics the in vivo degranulation process, which occurs upon allergen exposure in sensitized patients or in skin prick tests. Additionally, these assays are usually employed to investigate the allergenic potential of proteins and the reactivity of patients' sera's reactivity. Herein, we describe a simple 2-day protocol using an immortalized rat basophil leukemia cell line transfected and humanized with the human high-affinity IgE plasma-membrane receptor (Fc&#949;RI). This variant of the mediator release assay is a robust, sensitive, and reproducible in vitro cell-based system without the need to immobilize the antigen to solid matrices. The protocol consists of the following steps: (1) complement inactivation of human sera, (2) harvesting, seeding, and passive sensitization of the cells, (3) stimulation with antigen to cause mediator release, and (4) measuring of &#946;-hexosaminidase activity as a surrogate for the released inflammatory mediators, such as histamine. The assay represents a useful tool to assess the capacity of the allergen-IgE cross-linking to trigger cell degranulation and can be implemented to standardize allergen extracts, to compare patients' reactivity to minor or major allergens and to allergenic extracts (pollen, cat dander, etc.), to investigate the potency of allergen homologs, isoforms, and fold-variants (e.g., hypoallergenicity), as well as the effects of ligands on the allergenic activity. A more recent application includes the use of the assay to monitor the treatment efficacy in the course of allergen immunotherapy.

Here we present the mediator release assay, using a rat basophilic leukemia cell line transfected with the human IgE receptor, to simulate the degranulation of effector cells typically observed in type 1 allergic reactions. This method investigates the biological activity of allergens in a highly sensitive, reproducible, and tailorable manner.

Mediator release assays analyze in vitro immunoglobulin E (IgE)-mediated degranulation and secretion of mediators by effector cells, such as mast cells ...

Ligand Binding Sites

Proteins are dynamic macromolecules that carry out a wide variety of essential processes; however, the activities of most proteins depend on their interactions with other molecules or ions, known as ligands.
Protein-ligand interactions are quite specific; even though numerous potential ligands surround a cellular protein at any given time, only a particular ligand can bind to that protein. Moreover, a ligand binds only to a dedicated area on the surface of the protein, known as the ligand-binding site. The specificity of a protein&#8217;s ligand-binding site is determined by the arrangement of its amino acid chain which gives the area its shape and chemical reactivity. Hence, a ligand-binding site provides a complementary shape to its ligand and keeps the ligand in place via chemical interactions. These chemical interactions are often noncovalent; however, since these interactions are reversible and weak, many of these interactions need to occur simultaneously to hold the protein and the ligand together.
Research that elucidates interaction mechanisms at ligand binding sites generally involves in silico modeling and in vitro approaches. In silico modeling uses computers to compare previously known protein structures and evolutionary data to make predictions to determine the optimal binding shape and energy state of the protein-ligand complex. In vitro approaches compliment in silico predictions by providing evidence for ligand binding through binding and kinetic assays in the laboratory. Ligand binding research is important for understanding the functions of proteins and how they perform specific cellular processes in both healthy, as well as in diseased conditions. For instance, certain genetic conditions and cancers can alter the sequence of a protein, ultimately affecting its ability to bind a ligand. In addition, this research also allows scientists to design drugs with specific interactions and minimal side effects by targeting the ligand-binding site of an implicated protein.