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.
