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>

<ol>
	<li>Radauer, C., Bublin, M., Wagner, S., Mari, A., 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=Allergens+are+distributed+into+few+protein+families+and+possess+a+restricted+number+of+biochemical+functions.">Allergens are distributed into few protein families and possess a restricted number of biochemical functions.</a> Journal of Allergy and Clinical Immunology. 121 (4), 847-852 (2008).</li><li>Dearman, R. J., Alcocer, M. J. C., Kimber, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+plant+lipids+on+immune+responses+in+mice+to+the+major+Brazil+nut+allergen+Ber+e+1.">Influence of plant lipids on immune responses in mice to the major Brazil nut allergen Ber e 1.</a> Clinical and Experimental Allergy. 37 (4), 582-591 (2007).</li><li>Ichikawa, S., 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=Lipopolysaccharide+binding+of+the+mite+allergen+Der+f+2.">Lipopolysaccharide binding of the mite allergen Der f 2.</a> Genes to Cells. 14 (9), 1055-1065 (2009).</li><li>Mueller, G. 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=The+structure+of+the+dust+mite+allergen+Der+p+7+reveals+similarities+to+innate+immune+proteins.">The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins.</a> Journal of Allergy and Clinical Immunology. 125 (4), 909-917 (2010).</li><li>Reginald, K., Chew, F. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+allergen+Der+p+2+is+a+cholesterol+binding+protein.">The major allergen Der p 2 is a cholesterol binding protein.</a> Scientific Reports. 9 (1), 1556 (2019).</li><li>Trompette, 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=Allergenicity+resulting+from+functional+mimicry+of+a+Toll-like+receptor+complex+protein.">Allergenicity resulting from functional mimicry of a Toll-like receptor complex protein.</a> Nature. 457 (7229), 585-589 (2009).</li><li>Angelina, 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=The+lipid+interaction+capacity+of+Sin+a+2+and+Ara+h+1,+major+mustard+and+peanut+allergens+of+the+cupin+superfamily,+endorses+allergenicity.">The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity.</a> Allergy: European Journal of Allergy and Clinical Immunology. 71 (9), 1284-1294 (2016).</li><li>Soh, W. T., 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=Multiple+roles+of+Bet+v+1+ligands+in+allergen+stabilization+and+modulation+of+endosomal+protease+activity.">Multiple roles of Bet v 1 ligands in allergen stabilization and modulation of endosomal protease activity.</a> Allergy: European Journal of Allergy and Clinical Immunology. 74 (12), 2382-2393 (2019).</li><li>Foo, A. C. Y., 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=Hydrophobic+ligands+influence+the+structure,+stability,+and+processing+of+the+major+cockroach+allergen+Bla+g+1.">Hydrophobic ligands influence the structure, stability, and processing of the major cockroach allergen Bla g 1.</a> Scientific Reports. 9 (1), 18294 (2019).</li><li>Machado, Y., 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=Fold+Stability+is+a+key+factor+for+immunogenicity+and+allergenicity+of+the+major+birch+pollen+allergen+Bet+v1.0101.">Fold Stability is a key factor for immunogenicity and allergenicity of the major birch pollen allergen Bet v1.0101.</a> Allergy: European Journal of Allergy and Clinical Immunology. 137 (5), 1525-1534 (2016).</li><li>Mueller, G. 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=The+novel+structure+of+the+cockroach+allergen+Bla+g+1+has+implications+for+allergenicity+and+exposure+assessment.">The novel structure of the cockroach allergen Bla g 1 has implications for allergenicity and exposure assessment.</a> Journal of Allergy and Clinical Immunology. 132 (6), (2013).</li><li>Mogensen, J. E., Wimmer, R., Larsen, J. N., Spangfort, M. D., Otzen, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+major+birch+allergen+,+Bet+v+1+,+shows+affinity+for+a+broad+spectrum+of+physiological+ligands.">The major birch allergen , Bet v 1 , shows affinity for a broad spectrum of physiological ligands.</a> The Journal of Biological Chemistry. 277 (26), 23684-23692 (2002).</li><li>Seutter von Loetzen, 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=Secret+of+the+major+birch+pollen+allergen+Bet+v+1:+identification+of+the+physiological+ligand.">Secret of the major birch pollen allergen Bet v 1: identification of the physiological ligand.</a> Biochemical Journal. 457 (3), 379-390 (2014).</li><li>Ib&#225;&#241;ez-Shimabukuro, 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=Structure+and+ligand+binding+of+As-p18,+an+extracellular+fatty+acid+binding+protein+from+the+eggs+of+a+parasitic+nematode.">Structure and ligand binding of As-p18, an extracellular fatty acid binding protein from the eggs of a parasitic nematode.</a> Bioscience Reports. 39 (7), 1-16 (2019).</li><li>Beyer, K., Klingenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ADP/ATP+carrier+protein+from+beef+heart+mitochondria+has+high+amounts+of+tightly+bound+cardiolipin,+as+revealed+by+31P+nuclear+magnetic+resonance.">ADP/ATP carrier protein from beef heart mitochondria has high amounts of tightly bound cardiolipin, as revealed by 31P nuclear magnetic resonance.</a> Biochemistry. 24 (15), 3821-3826 (1985).</li><li>Delaglio, F., 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=Nmrpipe+-+a+multidimensional+spectral+processing+system+based+on+unix+pipes.">Nmrpipe - a multidimensional spectral processing system based on unix pipes.</a> Journal of Biomolecular NMR. 6 (3), 277-293 (1995).</li><li>Johnson, B. A., Blevins, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+View:+A+computer+program+for+the+visualization+and+analysis+of+NMR+data.">NMR View: A computer program for the visualization and analysis of NMR data.</a> Journal of Biomolecular NMR. 4 (5), 603-614 (1994).</li><li>Dillon, M. B. 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=Different+Bla-g+T+cell+antigens+dominate+responses+in+asthma+versus+rhinitis+subjects.">Different Bla-g T cell antigens dominate responses in asthma versus rhinitis subjects.</a> Clinical and Experimental Allergy. 45, 1856-1867 (2015).</li><li>Pasquato, 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=Crystal+structure+of+peach+Pru+p+3,+the+prototypic+member+of+the+family+of+plant+non-specific+lipid+transfer+protein+pan-allergens.">Crystal structure of peach Pru p 3, the prototypic member of the family of plant non-specific lipid transfer protein pan-allergens.</a> Journal of Molecular Biology. 356 (3), 684-694 (2006).</li><li>Dubiela, P., 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=Impact+of+lipid+binding+on+the+tertiary+structure+and+allergenic+potential+of+Jug+r+3,+the+non-specific+lipid+transfer+protein+from+walnut.">Impact of lipid binding on the tertiary structure and allergenic potential of Jug r 3, the non-specific lipid transfer protein from walnut.</a> Scientific Reports. 9 (2007), 1-11 (2019).</li><li>Abdullah, S. 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=Ligand+binding+to+an+allergenic+lipid+transfer+protein+enhances+conformational+flexibility+resulting+in+an+increase+in+susceptibility+to+gastroduodenal+proteolysis.">Ligand binding to an allergenic lipid transfer protein enhances conformational flexibility resulting in an increase in susceptibility to gastroduodenal proteolysis.</a> Scientific Reports. 6, 30279 (2016).</li><li>Derewenda, 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=The+crystal+structure+of+a+major+dust+mite+allergen+Der+p+2+,+and+its+biological+implications.">The crystal structure of a major dust mite allergen Der p 2 , and its biological implications.</a> Journal of Molecular Biology. 318 (1), 189-197 (2002).</li><li>Lipfert, J., Columbus, L., Chu, V. B., Lesley, S. A., Doniach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+and+shape+of+detergent+micelles+determined+by+small-angle+X-ray+scattering.">Size and shape of detergent micelles determined by small-angle X-ray scattering.</a> The Journal of Physical Chemistry. B. 111 (43), 12427-12438 (2007).</li><li>Pulsawat, P., 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=The+house+dust+mite+allergen+Der+p+5+binds+lipid+ligands+and+stimulates+airway+epithelial+cells+through+a+TLR2-dependent+pathway.">The house dust mite allergen Der p 5 binds lipid ligands and stimulates airway epithelial cells through a TLR2-dependent pathway.</a> Clinical and Experimental Allergy. 49 (3), 378-390 (2019).</li><li>Douliez, J. P., Michon, T., Marion, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steady-state+tyrosine+fluorescence+to+study+the+lipid-binding+properties+of+a+wheat+non-specific+lipid-transfer+protein+(nsLTP1).">Steady-state tyrosine fluorescence to study the lipid-binding properties of a wheat non-specific lipid-transfer protein (nsLTP1).</a> Biochimica et Biophysica Acta - Biomembranes. 1467 (1), 65-72 (2000).</li><li>Ogburn, R. 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 their endogenously-bound cargoes when purified using standard affinity and size-exclusion chromatography methods13,19,20,21,22. These unwelcome guests could alter the biophysical and immunological properties of these proteins in a similar manner, highlighting the need for techniques to ensure complete delipidation such as the one presented here.
While the use of reverse-phase HPLC in the purification of allergens has been described previously2, coupling it with a thermal annealing protocol provides the rather unusual opportunity to reconstitute allergens with a range of natural and un-natural ligands, allowing users to probe lipid-allergen interactions. This thermal denaturation step was found to be essential for two main purposes. First, thermal denaturation is required to facilitate ligand access to their binding cavities which, due to their hydrophobic nature, are often buried away from the aqueous solvent9,22. Secondly, hydrophobic ligands such as fatty acids and phospholipids often form larger supramolecular structures such as micelles or vesicles when placed in an aqueous environment. The concentration of monomeric, or &#8220;free&#8221; ligands available for protein binding can be approximated using the critical micelle concentration (CMC). DSPC and other long-chain phospholipids have CMC values in the nM range, indicating that there are virtually no free ligands available for Bla g 1 binding. Even short chain lipids and fatty acids have CMC&#8217;s in the low &#181;m to mM range, indicating that a large proportion of these ligands remain in the micellar or bilayer phase23. However, the high temperatures employed in our denaturation protocol disperses these larger aggregates, facilitating binding. Previous studies have typically employed prolonged incubation periods to facilitate this process. However, the lack of a thermal denaturation/annealing process raises doubts to the efficacy of loading. For example, incubating the mite allergen Der p 5 with the fluorescent fatty acid analogue 11-(Dansylamino)undecanoic acid (DAUDA) yielded a binding stoichiometry of 0.66 despite possessing a large hydrophobic cavity on par with Bla g 124. Likewise, the binding specificity and stoichiometry of plant nsLTPs were found to vary greatly depending on whether the lipids and protein are first solubilized in methanol prior to the addition of aqueous buffer, indicating that ligand and/or binding site accessibility was a limiting factor25.
In addition to Bla g 1, we have successfully applied the same strategy to several other MA domain proteins from cockroaches and mosquito (A. aegypti), as well as Der p 2 (data not shown). We noted that both the Bla g 1 homologues and Der p 2 eluted at a different time than Bla g 1 from the C18 column (step 3.3). The elution gradients in this step may need to be optimized for other proteins.&#160; Alternatively, HPLC columns with a less hydrophobic stationary phase (e.g., C8) may be employed, though in the case of Bla g 1 the increased hydrophobicity of the C18 column was necessary to completely remove diacyl phospholipid contaminants from ecBla g 1. Despite the differences in biophysical and biochemical properties, we have found this protocol to be extremely robust and could be easily applied to other allergenic proteins. While the harsh conditions employed may present a potential limitation, the increased resilience observed for many allergens reduces its impact26,27. Indeed, several food and inhalation allergens such as Der p 2, Ber e 1, Ara a 6 and Lep w 1 have been observed to recover their structure and immunogenicity following thermal denaturation, though optimization of buffer conditions may be required28,29,30,31,32,33; for example reversible denaturation of nsLTP&#8217;s (Cor a 8) and thaumatins (Mal d 2 and Act d 2) is only observed under acidic (pH &#60;4) conditions28,30,31. Additionally, it should be noted that the authors did not attempt to optimize either the timing or temperatures employed in the annealing protocol. It is possible that ligand solubilization and protein folding/unfolding may be achieved using a lower maximum temperature as seen with Ber e 1 for which reversible denaturation is achieved at 82 &#730;C29. The use of such measures is expected to expand the range of allergens to which this protocol can be applied. &#160;
Another important consideration when adapting this protocol to other allergen systems is the concentration of ligands required during the annealing process. In the case of Bla g 1 the expected yield is ~0.25&#8211;0.4 &#956;mol of protein per 1 L cell culture. Given the demonstrated binding stoichiometry of 8 fatty acids or 4 diacyl chain lipids per allergen, a 20&#8211;40 fold molar excess of cargo (5&#8211;10 &#956;mol) was employed. It should be noted that the lipid binding ability of Bla g 1 and its homologues is unique; for example nsLTP&#8217;s are generally accepted to bind at most two lipid ligands25 while lipocalins have less than 1 stoichiometry34. As such, complete loading of these types of allergens may be accomplished with a smaller excess of ligands. A final consideration when adapting this protocol to other allergen systems is the presence of disulfide bonds, which can be problematic if not properly formed prior to denaturing. One possible approach would be to carry out the annealing process in the presence of a reducing agent such as 2 mM DTT. The native disulfide bonds could be subsequently re-formed through the addition of reduced and oxidized glutathione as described for the peanut allergen fragment studied by Aalberse et al.35. In this case, recovery of the correct disulfide bonding should be empirically assessed by mass spectrometry35.
In this work we describe a technique through which allergens can be delipidated and re-annealed with various phospholipid and fatty acid cargoes. However, there are many other classes of potentially immunogenic or adjuventising ligands present within common allergen reservoirs. For example, cat, dog, and mite allergens have been proposed to bind lipopolysaccharides (LPS) and other bacterial lipids from house dust36, while the Bet v 1 has been shown to extract complex flavonoids from the pollen matrix13. The protocol described in this work can be easily adapted to explore the role of these lipids in a more detailed manner. As a proof of concept we have been able to demonstrate that the hydrophobic cavity of Bla g 1 is capable of binding lipoteichoic acid (LTA) from the cell walls of gram positive bacteria, but excludes LPS from gram negative species, potentially reflecting the greater number of acyl chains in the latter9. Taking this one step further, one could utilize the thermal denaturation/annealing protocol to incorporate fluorescent probes and other non-natural fatty acid analogues into allergen proteins. Indeed, we were able to load the hydrophobic cavity of the mosquito homologue of Bla g 1 with DAUDA, opening additional avenues to examine the effects of lipid ligands on allergenic disease.

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bla g 1 Gene&#160;</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-&#946;-D-thiogalactopyranoside (IPTG)</td>
			<td>Fisher Scientific</td>
			<td>34060</td>
			<td></td>
		</tr>
		<tr>
			<td>Jasco&#160; 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.&#160;</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&#8211;293 (1995).</td>
		</tr>
		<tr>
			<td>NMRViewJ (Software)</td>
			<td>Johnson et al.&#160;</td>
			<td>N/a</td>
			<td>Johnson, B. A. &#38; Blevins, R. A. NMR View: A computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603&#8211;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 cycles of sample loading and elution could provide remedy for this issue.
Applying the Bla g 1 to a reverse-phase C18 column yields a distinctive elution profile (Figure 1B), with two large peaks at ~50% buffer B, and a second large peak at ~75% buffer B. SDS-PAGE analysis of the resulting fractions suggest that the former correspond to the cleaved GST tag, while the latter corresponds to Bla g 1. Occasionally a third, smaller peak will occur in the middle corresponding to residual, un-cleaved GST-Bla g 1. The presence of this un-cleaved product can be eliminated by increasing the amount of TEV employed in the cleavage reaction or extending the incubation time. While incomplete cleavage will reduce the yield, the separation obtained on the C18 column is sufficient to ensure that the purity of the final Bla g 1 product remains uncompromised. A consequence of reverse-phase HPLC is that the final protein product is eluted into an organic solvent environment. While this facilitates removal of any hydrophobic ligands, removal of this solvent via lyophilization is required, yielding a fluffy white powder (Figure 1C).
Annealing of the protein is required to reconstitute the native Bla g 1 fold and can be carried out either in the absence or presence of a lipid cargo. Addition of DMSO to the dried Bla g 1 and phospholipid cargoes prior to the refolding buffer facilitates the solubilization process, though some longer chain lipid cargoes will not fully dissolve even at elevated temperatures. However, this was not observed to impact the loading efficacy among the lipids tested in our studies (Figure 1C). Similarly, excess lipids will often precipitate out of solution or form large vesicles upon cooling, resulting in a cloudy appearance after annealing (Figure 1C). This was also not observed to effect loading efficiency, and any aggregates are readily removed through the filtration and subsequent buffer exchange steps to yield a clear, transparent solution. Despite the harsh conditions, no thermolysis was observed for Bla g 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61780/61780fig1.jpg" /> 
Figure 1: Initial purification of Bla g 1. (A) SDS-PAGE showing the soluble protein fraction following initial lysis (S); flow-through (FT), wash (W), and elution from the glutathione-sepharose column (E); and the final Bla g 1 product following TEV cleavage of the GST tag (TEV). The HPLC elution profile of the resulting Bla g 1 product following TEV cleavage is shown in (B). A280 is shown in blue, while the elution gradient (% Buffer B) is shown in green. Fractions corresponding to the cleaved GST tag (H1, H2), residual un-cleaved GST-Bla g 1 (H3), and purified Bla g 1 (H4) are indicated with red arrows at ~50%, ~65%, and ~74% Buffer B respectively. SDS-PAGE analysis of fractions H1- H4 are shown in (A) and labeled accordingly. (C) Representative images showing Bla g 1 at various stages of the annealing process. Note that the precise and extent of precipitate formation as depicted in ii and iii is dependent on the type of lipid cargo employed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61780/61780fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
31P-NMR spectra of Apo-Bla g 1 purified in this manner show no detectable phospholipids either by NMR (Figure 2A) or thin layer chromotography (data not shown). By contrast, similar spectra obtained for Bla g 1 loaded with a distearoylphosphatidylcholine (DSPC) phospholipid show a strong peak corresponding to the phosphatidylcholine headgroup. For comparison, a representative 31P-NMR spectrum of Bla g 1 purified from recombinant E. coli without the use of the lipid removal/annealing protocol described herein (ecBla g 1) show a heterogeneous mixture of endogenous lipids extracted from the recombinant expression system (Figure 2B). Taking advantage of the quantitative nature of NMR, a standard curve can be produced using reference samples of known DSPC concentrations (Figure 2C). Comparing the 31P signal intensity obtained from DSPC-Bla g 1 against this standard curve yields a binding stoichiometry of 4.7 &#177; 0.5 lipids per protein; a value that compares favorably to the predicted full binding stoichiometry obtained from in silico&#160; studies and structural analysis9. Note that this technique will only detect ligands which contain a 31P nucleus such as phospholipids, lysophospholipids, lipopolysaccharides etc. However, this protocol can be easily adapted for 13C-NMR analysis. In this case, methyl-13C labeled fatty acids would be recommended due to its favorable NMR relaxation properties. Restricting isotopic labeling to a single site also facilitates spectral interpretation, as only a single peak is expected, while simultaneously reducing the cost relative to uniform 13C-labeled counterparts. An alternative approach would be to employ mass-spec to identify bound ligands, as demonstrated in a previous study which identified a mixture of fatty acids as the natural cargo of Bla g 1 isolated from cockroach frass (nBla g 1)9. However, the limited quantitation capabilities of mass spec precluded an accurate measurement of binding stoichiometry without sufficient standards.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61780/61780fig2.jpg" /> 
Figure 2: Verifying lipid removal and loading of Bla g 1. (A) 31P-NMR spectra of Apo- (black) or DSPC-loaded Bla g 1 (red) prepared using the annealing protocol described in this work demonstrating the complete removal of lipids in the former, and the homogeneous loading of phosphatidylcholine (PC) lipids achieved in the latter. In contrast, Bla g 1 purified from recombinant E. coli without lipid stripping and annealing (ecBla g 1) shows a heterogeneous mixture of endogenous phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) lipids when analyzed using this method (B). A representative standard curve obtained from DSPC reference samples of known concentrations is shown in (C), from which the Bla g 1 binding stoichiometry can be obtained. Figures adapted from Foo et al. (2019) and presented under the Creative Commons CC BY License9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61780/61780fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Crystal structures of Bla g 1 reveal a unique fold consisting of 12 amphipathic alpha-helices. Circular dichroism represents a quick and convenient method to assess whether this fold has been successfully reconstituted after the annealing process. CD spectra for Apo- and lipid (nMix)-loaded Bla g 1 show minima ~220 and 210 nm indicative of a predominantly alpha-helical structure (Figure 3A). This spectrum is extremely similar to that obtained for ecBla g 1 and nBla g 1, providing further evidence that the native structure of Bla g 1 is successfully recovered. This was further confirmed through the use of 19F and 1H-15N solution-NMR, a full discussion of which is available elsewhere9. CD-based thermal denaturation assays show a cooperative loss of alpha-helical secondary structure indicative of a folded globular domain (Figure 3B). Analysis of the resulting melting temperatures (Figure 3C) show a significant increase upon nMix ligand binding. This elevated thermostability is in line with that calculated for nBla g 1, indicating that we are able to fully reproduce the natural state of Bla g 1. Note that ecBla g 1 also shows a similar, if not greater enhancement in thermostability, illustrating the potential for residual endogenously bound lipids to interfere with biophysical characterization of allergens purified using traditional FPLC-based approaches. In contrast, the ability to quantitatively remove and reload hydrophobic cargoes from allergens such as Bla g 1 provides a unique avenue to examine the role of lipids in the allergic response. Here, we describe a method to examine the influence of lipid cargoes on the structure, stability, and endosomal processing of the allergenic proteins themselves, though other avenues of study could be considered.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61780/61780fig3.jpg" /> 
Figure 3: Confirming successful recovery of the Bla g 1 fold. (A) CD spectra of Apo- (black) or nMix-loaded (red) Bla g 1 purified and annealed using the protocol described herein, with minima at ~220 and 210 nm indicative of a predominantly alpha-helical structure consistent with the available X-ray crystal structure. Both Apo- and nMix-loaded Bla g 1 spectra are extremely similar with that obtained for Bla g 1 purified from recombinant E. coli (ecBla g 1, green) or from its natural allergenic source (nBla g 1, blue) without the lipid removal and annealing protocol, further supporting the successful recovery of the native structure in the former. (B) Representative thermal profiles for Apo- (black) and nMix-loaded (red) Bla g 1 showing a sigmoidal curve indicative cooperative unfolding. nBla g 1 (blue) and ecBla g 1 (green) shown as reference. The calculated melting temperatures (MT25) of Bla g 1 are shown in (C). Binding of nMix ligands (red) yields a significant increase in thermostability relative to Apo-Bla g 1 (black). This mirrors the trend observed for nBla g 1 (blue), suggesting that we are able to successfully recover the native state. The even greater stability observed for ecBla g 1 highlights the potential of endogenously bound lipids to interfere with biophysical characterization of allergens. MT25 values presented in C represent the mean value obtained from at least three independent trials. Error bars represent the corresponding standard deviation values. Figures adapted from Foo et al. (2019) and presented under the Creative Commons CC BY License9. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61780/61780fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

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

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding interactions. This information is critical in the design of new pharmaceutical compounds. However, many pharmaceutically relevant ligands are chemically unstable at the high temperatures used in DSC analyses. Thus, measuring binding interactions is challenging because the concentrations of ligands and thermally-converted products are constantly changing within the calorimeter cell. Here, we present a protocol using thermolabile ligands and DSC for rapidly obtaining thermodynamic and kinetic information on the folding, binding, and ligand conversion processes. We have applied our method to the DNA aptamer MN4 that binds to the thermolabile ligand cocaine. Using a new global fitting analysis that accounts for thermolabile ligand conversion, the complete set of folding and binding parameters are obtained from a pair of DSC experiments. In addition, we show that the rate constant for thermolabile ligand conversion may be obtained with only one supplementary DSC dataset. The guidelines for identifying and analyzing data from several more complicated scenarios are presented, including irreversible aggregation of the biomolecule, slow folding, slow binding, and rapid depletion of the thermolabile ligand.

We present a protocol for rapid characterization of biomolecular folding and binding interactions with thermolabile ligands using differential scanning calorimetry.

Differential scanning calorimetry (DSC) is a powerful technique for quantifying thermodynamic parameters governing biomolecular folding and binding ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

Looking Outwards: Isolation of Cyanobacterial Released Carbohydrate Polymers and Proteins

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The identification and characterization of these biomolecules can improve knowledge about their secretion pathways and help to manipulate them. Furthermore, some of these biomolecules are also interesting in terms of biotechnological applications. Described here are two protocols for easy and rapid isolation of cyanobacterial released carbohydrate polymers and proteins. The method for isolation of released carbohydrate polymers is based on conventional precipitation techniques of polysaccharides in aqueous solutions using organic solvents. This method preserves the characteristics of the polymer and simultaneously avoids the presence of contaminants from cell debris and culture medium. At the end of the process, the lyophilized polymer is ready to be used or characterized or can be subjected to further rounds of purification, depending on the final intended use. Regarding the isolation of the cyanobacterial exoproteome, the technique is based on the concentration of the cell-free medium after removal of the major contaminants by centrifugation and filtration. This strategy allows for reliable isolation of proteins that reach the extracellular milieu via membrane transporters or outer membrane vesicles. These proteins can be subsequently identified using standard mass spectrometry techniques. The protocols presented here can be applied not only to a wide range of cyanobacteria, but also to other bacterial strains. Furthermore, these procedures can be easily tailored according to the final use of the products, purity degree required, and bacterial strain.

Here, protocols for the isolation of cyanobacterial released carbohydrate polymers and isolation of their exoproteomes are described. Both procedures embody key steps to obtain polymers or proteins with high purity degrees that can be used for further analysis or applications. They can also be easily adapted according to specific user needs.

Cyanobacteria can actively secrete a wide range of biomolecules into the extracellular environment, such as heteropolysaccharides and proteins. The ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

Imaging of Podocytic Proteins Nephrin, Actin, and Podocin with Expansion Microscopy

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte foot processes contain actin bundles that bind to cytoskeletal adaptor proteins such as podocin. Those adaptor proteins, such as podocin, link the backbone of the glomerular slit diaphragm, such as nephrin, to the actin cytoskeleton. Studying the localization and function of these and other podocytic proteins is essential for the understanding of the glomerular filter&#39;s role in health and disease. The presented protocol enables the user to visualize actin, podocin, and nephrin in cells with super resolution imaging on a conventional microscope. First, cells are stained with a conventional immunofluorescence technique. All proteins within the sample are then covalently anchored to a swellable hydrogel. Through digestion with proteinase K, structural proteins are cleaved allowing isotropical swelling of the gel in the last step. Dialysis of the sample in water results in a 4-4.5-fold expansion of the sample and the sample can be imaged via a conventional fluorescence microscope, rendering a potential resolution of 70 nm.

The presented method enables visualization of fluorescently labeled cellular proteins with expansion microscopy leading to a resolution of 70 nm on a conventional microscope.

Disruption of the glomerular filter composed of the glomerular endothelium, glomerular basement membrane and podocytes, results in albuminuria. Podocyte ...

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay (DRaCALA)

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins of several nucleotide-derived secondary messengers (NSMs) have been systematically identified and studied in model organisms. These achievements are mainly due to the development of several new techniques including the capture compound technique and the differential radial capillary action of ligand assay (DRaCALA), which were used to systematically identify target proteins of these small molecules. This paper describes the use of the NSMs, guanosine penta- and tetraphosphates (p)ppGpp, as an example and video demonstration of the DRaCALA technique. Using DRaCALA, 9 out of 20 known and 12 new target proteins of (p)ppGpp were identified in the model organism, Escherichia coli K-12, demonstrating the power of this assay. In principle, DRaCALA could be used for studying small ligands that can be labeled by radioactive isotopes or fluorescent dyes. The critical steps, pros, and cons of DRaCALA are discussed here for further application of this technique.

Identifying the Binding Proteins of Small Ligands with the Differential Radial Capillary Action of Ligand Assay DRaCALA

The Differential Radial Capillary Action of Ligand Assay (DRaCALA) can be used to identify small ligand binding proteins of an organism by using an ORFeome library.

The past decade has seen tremendous progress in the understanding of small signaling molecules in bacterial physiology. In particular, the target proteins ...

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...