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 direct....

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 ins.......

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

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