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

Summary

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.

Abstract

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.

Introduction

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 proteins^3,4,5. 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 process^5,6. It is also possible that endogenously bound lipids could alter the biophysical properties of allergenic 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 work^9,10.

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 cavity^9,11. 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 “natural” ligands^9,11. 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 1^12,13. 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 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.

Protocol

1. Bla g 1 cloning

1. 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.
2. 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.
3. Transform the Bla g 1 pGEX vector into BL21 DE3 E. coli cells.
   1. Prepare a 10 ng/µL stock of the desired vector.
   2. Combine 1 µL of 10 ng/µL DNA stock with 50 µL of BL21 DE3 cells as provided by the manufacturer.
   3. Incubate BL21 DE3-DNA mixture for 30 min on ice. Transfer to a 42 ˚C water bath for 1 min, then immediately transfer back on ice for an additional 1 min incubation.
   4. Add 200 µL of LB media to the cells and incubate for an additional 1 h at 37 ˚C.
   5. Plate the transformed cells on LB-Agar plates containing 100 mg/L ampicillin and grow at 37 ˚C overnight.

2. Initial expression and purification

1. 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 ˚C overnight.
2. 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 ˚C to an OD₆₀₀ > 0.6.
3. Induce protein expression through the addition of 0.5 mM IPTG. Transfer cells to 18 ˚C and incubate overnight.
4. On the next day, harvest cells as described in 2.2. The resulting cell pellet can be frozen and stored at -20 ˚C.
5. 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 µL of benzonase nuclease.
6. Lyse cells using a probe sonicator (500 W, 20 kHz) set to 30–50% power for 4 min with a 50% duty cycle. Keep the lysate in an ice bath during sonication
7. Centrifuge lysate at 45,000 x g for 20 min. Discard insoluble fraction (pellet).
   1. Remove 28 µL of soluble protein. Combine with 7 µL 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.
8. Apply soluble proteins (supernatant) to a glutathione resin column (~10 mL total bed volume) equilibrated in PBS pH 7.4.
9. Wash out any unbound proteins using 50 mL of PBS.
10. Elute GST-Bla g 1 using 50 mL of PBS containing 10 mM reduced glutathione.
11. Incubate eluted protein with 0.2 kU TEV protease overnight at 4 ˚C, or room temperature for 6 h to remove GST tag.

3. Endogenous lipid removal via reverse-phase HPLC

1. Collect the cleaved Bla g 1 and concentrate it to ~2 mL using a centrifugal filter unit with a <10 kDa molecular weight cut-off.
   1. Add <12 mL sample to the top of concentrator and spin at 5,000 x g for 10–15 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.
2. 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 < 5 μm and pore size of >200 Å 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).
3. Elute Bla g 1 using the protocol shown in Table 1 at a flow rate of 1.5–4.0 mL/min. Monitor the elution process using the fluorescence absorbance at 280 nm.
   1. Collect and pool Bla g 1 fractions. Bla g 1 normally elutes at >74% buffer B, or ~34–40 min.
      NOTE: Elution time will vary slightly depending on the flow rate or column size. Collect fractions based on A₂₈₀ for best results.

Time (Min)	Buffer A (%)	Buffer B (%)
0	97	3
10	97	3
25	35	65
55	5	95
65	5	95
70	97	3

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.

4. 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.
   1. Prepare a separate 1 mL aliquot (test aliquot). This will be used to determine the expected yield.
5. Freeze the samples and test aliquot by placing them in a -80 ˚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.
6. Dry the resulting delipidated protein samples using a lyophilizer. Dried protein may be stored at 4 ˚C for several months in a sealed container.

4. Reconstitution of Apo- and cargo-loaded Bla g 1

1. Determine the anticipated Bla g 1 yield.
   1. Resuspend lyophilized, delipidated (post-HPLC) test aliquot in 5 mL of refolding buffer, (50 mM HEPES pH 7.4, 100 mM NaCl, 2% DMSO).
   2. Heat the mixture in a water bath (500 mL beaker with 250 mL water and stir bar over a hot plate) to 95 ˚C. Vortex solutions intermittently and incubate at 95 ˚C for 0.5–1 h.
   3. 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 ˚C if needed.
   4. Pass annealed Bla g 1-lipid mixture through a 0.22 µM syringe filter to remove particulate matter.
   5. 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.
   6. 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.
2. Reconstitute Apo-  or cargo-loaded Bla g 1
   1. Resuspend Bla g 1 aliquots in refolding buffer as described in 4.1.1.
   2. To produce Apo-Bla g 1, repeat steps 4.1.2–4.1.6 to obtain desired yield.
   3. To load Bla g 1 with fatty acids, prepare 20 mM stock solutions of the desired fatty acid cargo in methanol or DMSO.  Then, skip to step 4.2.5.
   4. To load Bla g 1 with phospholipids, prepare a 10 mg/mL stock of the desired cargo in chloroform inside a glass test tube.
      1. 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.
      2. 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.
      3. 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 10–20% 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.
   5. 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 ~0.25–0.4 nmol protein, corresponding to ~400 µM ligand per tube.
   6. Anneal the protein as described in 4.1.

5. Confirming phospholipid cargo removal/loading via ³¹P-NMR

1. Concentrate samples of Apo- or cargo-loaded Bla g 1 to >100 µM using a centrifugal filter unit as described in 3.1.
2. Rehydrate reference phospholipid in PBS buffer to final concentrations of 2, 1.5, 1, 0.5, and 0.25 mM.
3. 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 µL.
   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  chloroform/methanol as described previously¹⁵.
4. 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.
5. Process the resulting data using appropriate software¹⁶.
6. Obtain peak intensities using preferred NMR viewing software¹⁷.
7. 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).
   1. Confirm full binding stoichiometry by comparing the peak intensity of the Bla g 1 spectrum to that of the phospholipid reference standards.

6. Confirming Bla g 1 folding

1. Prepare 0.5 µM samples of Bla g 1 in CD buffer (100 mM KH₂PO₄, buffer pH 7.5). Load 2 mL of the sample into a 10 mm CD cuvette with magnetic stir bar.
2. Measure CD spectrum of Bla g 1 to confirm reconstitution of secondary structure. Ensure that Photomultiplier (PMT) voltage does not exceed manufacturer recommendations (generally 1 kV).
   1. Measure CD signal from 260–200 nm at 25 ˚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.
3. Increase the temperature in the CD cell from 25 ˚C–95 ˚C at a rate of 0.5 ˚C/min. Activate magnetic stir bar to ensure temperature is uniform across the sample.
4. Monitor CD at 222 nm, taking readings every 2 ˚C.
5. 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.

Results

Using affinity chromatography, recombinant GST-Bla g 1 was readily isolated to a high level of purity (Figure 1A), producing a yield of ~2–4 mg/L of cell culture. Overnight incubation with TEV protease at 4 ˚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 sam...

Discussion

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 immunogenicity^9,18. In addition to Bla g 1, other allergens such as Pru p 3 and Bet v 1 have been shown to retain the...

Materials

List of materials used in this article

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