Name: removal and replacement of endogenous ligands from lipid-bound proteins and allergens
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Description: Watch this Scientific Journal Video about Removal and Replacement of Endogenous Ligands from Lipid-Bound Proteins and Allergens at JoVE.com

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 ins...

<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. 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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. 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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 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 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 ...

Many allergens bind hydrophobic molecules. This protocol enables the complete removal and replacement of these ligands, allowing us to study their impact on structure and immunogenicity in a systematic manner. The use of reverse-phase HPLC, coupled with thermal annealing, has two advantages.It removes endogenous ligands, and it helps to solubilize ligands to open up otherwise-inaccessible binding sites. If we can determine the factors that contribute to allergenicity, we may be able to design therapies that avoid these factors. Many proteins bind lipid ligands, which are strongly retained and may influence both structure and function.Our method allows the systematic study of these interactions. The limited solubility and accessibility of many lipid cargoes may inhibit the loading process. Be sure to take care of when preparing these ligands, and to consider the need for system-specific adaptations.As working with hydrophobic and insoluble ligands is an adjustment for some allergists and biochemists, it can be useful to see what the samples look like at different stages. After cloning expression and purification, apply 12 milliliters of the cleaved Bla g 1 to a centrifugal filter unit with a less than 10 kilodalton molecular weight cutoff for multiple centrifugations in a swing bucket rotor until the total volume has been reduced to less than two milliliters. Load the resulting concentrate onto a 250 by 10 millimeter HPLC system equipped with a C18 reverse-phase chromatography column equilibrated with 97%buffer A and 3%buffer B.Elute Bla g 1 at a 1.5 to 4 milliliter per minute flow rate, as indicated in the table, using the fluorescence absorbance at 280 nanometers to monitor the elution process.Starting at around 34 to 40 minutes and a buffer B concentration above 74%collect no more than four milliliters of each Bla g 1 fraction. Aliquot the pooled Bla g 1 fraction into glass test tubes. Cover the tubes with paraffin film and perforate the film with two holes.Then, freeze the samples at minus 80 degrees Celsius for one hour and use a lyophilizer to dry the resulting de-lipidated protein samples. To determine the anticipated Bla g 1 yield, re-suspend a lyophilized de-lipidated test aliquot in five milliliters of refolding buffer. Heat the mixture in a 500-milliliter beaker containing 250 milliliters of water to 95 degrees Celsius on a hot plate with stirring and intermittent vortexing.Hold the solution at 95 degrees for 30 to 60 minutes before allowing the water bath to slowly equilibrate to room temperature. When the solution has cooled, pass the annealed Bla g 1 lipid mixture through a 0.22 micron syringe filter to remove particulate matter, and use a new centrifugal filter with a 10 kilodalton cutoff to buffer exchange the filtered protein three times into PBS to remove any residual free fatty acids and organic solvent. Then, assess the protein concentration using a standard protein analysis assay to determine the anticipated yield for the remaining Bla g 1 aliquots.To reconstitute the Apo-Bla g 1, re-suspend the Bla g 1 aliquots in five milliliters of refolding buffer per aliquot, and anneal the samples as demonstrated. To load the Bla g 1 with phospholipids, add 10 milligrams of the desired cargo to a glass test tube and dissolve in chloroform, and evaporate the chloroform to produce a lipid film. Then, add PBS to the tube to produce a final phospholipid concentration of 20 millimolar.Heat the phospholipid above the phase transition temperature of the lipid cargo to rehydrate the lipid film, and vortex until the solution turns cloudy. Then, add the phospho lipid cargo to produce a 20X molar excess of ligands relative to Bla g 1 based on the anticipated yield. A precipitant may form.Vortex to mix before annealing the protein as demonstrated. To confirm cargo removal or loading by phosphorus-31 NMR, use a centrifugal filter to concentrate the sample to greater than 100 micromolar as demonstrated. Prepare reference phospholipid samples of known concentrations in PBS buffer as indicated, and dilute the Bla g 1 end reference to a 1:1 ratio with cholate buffer to a total volume of about 600 microliters.Use a broadband probe to acquire one-dimensional, 31-phosphorus NMR spectra of the collate solubilized Bla g 1 samples and reference phospholipid standards, and compare the Bla g 1 31 phosphorous NMR spectra to those obtained for phospholipid reference samples to confirm the removal of endogenously bound ligands and/or the binding of the desired ligands based on the chemical shifts of the visible peaks, then compare the peak intensity of the Bla g 1 spectrum to that of the phospholipid reference standards to allow confirmation of the full binding stoichiometry. Using affinity chromatography, recombinant GST Bla g 1 can be readily isolated to a high level of purity, producing a yield of two to four milligrams per liter of cell culture. Overnight incubation with TEV protease at four degrees Celsius is sufficient to remove the GST tag, yielding the final product at approximately 24 kilodaltons.Applying the Bla g 1 to a reverse-phase C18 column yields a distinctive elution profile, with two large peaks at 50%buffer B, and a second large peak at 75%buffer B.Phosphorus-31 NMR spectra of Apo-Bla g 1 show no detectable phospholipids. A standard curve can be produced from the NMR using reference samples of known DSPC concentrations. Comparing the phosphorous 31 signal intensity obtained from DSPC Bla g 1 against this standard curve can be used to yield the binding stoichiometry of the lipids-per-protein.Circular dichroism spectra for Apo-and lipid-loaded Bla g 1 show minima of 220 and 210 nanometers, indicative of a predominantly alpha-helical structure. Circular dichroism-based thermal denaturation assays also show a co-operative loss of alpha-helical secondary structure, indicative of a folded globular domain. In addition, analysis of the resulting melting temperatures reveals a significant increase upon nMix ligand binding, consistent with Bla g 1 obtained from its natural allergen source.The success of this protocol relies on the ability to overcome both the limited solubility of hydrophobic ligands, and the inaccessible nature of the Bla g 1 binding cavity. This procedure lays the groundwork for immunological studies, such as T-cell proliferation assays, to assess the effect of lipid ligands on sensitization and the molecular mechanisms through which this occurs. Coupling this procedure with further biophysical assays, we have shown that the binding of hydrophobic ligands enhances Bla g 1 stability, with potential downstream implications for epitope generation and allergenicity.

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