Investigating the Almond allergen Pru du 4

Like other tree nuts, almonds can cause allergic reactions ranging from mild to life-threatening. For allergic individuals, experts still recommend avoiding exposure as the best way to manage allergies. To gain new insights into food allergies, researchers such as Zhang et al. are interested characterizing allergens. Profilins, the allergen inducing protein in almonds, are present in multiple plant species, but in very small amounts.This team of researchers isolated and expressed the almond profilin Pru du 4 using maltose binding protein (MBP) tag to enhance the expression and solubility.¹

To begin, the team collected almonds (Prunus dulcis) from a local orchard. They used extraction kits to elicit mRNA from ground almond kernels and synthesized cDNA from the total RNA. Next, they designed a set of PCR primers to amplify the Pru du 4 coding region and amplify the sumo coding sequence. After obtaining the final PCR product, the team inserted the sequence into the cloning vector pBluescript II SK with blunt-end ligation to make pBlue-SPrudu4. They transformed this ligation product into the E. coli strain dH5α. After sequencing the prepared plasmid from E. coli colonies to ensure correct insertion had occurred, they digested pBlue-SPrudu4 with BamHI and NcoI to release the sumo-Pru du 4 coding sequence. They performed another ligation to add pRSFdeut-1 vector to sumo-Pru du 4 to generate pRSF-SPrudu4. The team used another set of primers to amplify the MBP region and then inserted this sequence in the Bluescript II SK cloning vector.

After again confirming the correct MBP sequence, the team cut the plasmid with BspHI and BglII and ligated this product with the pRSF-SPrudu4 plasmid prepared earlier to generate the fusion protein pRSF1MS-SPrudu4. Next they transformed this product into into the E. coli strain BL21(DE3) for protein expression. After growing up the bacteria, they collected and lysed cells before purifying the fusion protein.

By digesting with sumo protease, the team was able to remove nearly 100% of the fusion protein without non-specific cleavage of the target protein or the fusion tag. From there, they analyzed the Pru du 4 protein using reverse phase high-powered liquid chromatography on an Easy nLC II and an Orbitrap Elite operated at 15,000 mass resolution (both from Thermo Scientific). The team used Excalibur and MagTran software to analyse and deconvolute the data across chromatographic peaks.

Zhang et al. was able to successfully express and isolate the Pru du 4 protein uniquely using sumo as a peptidase recognition site. The researchers stress the need for future experiments to understand the relationship between the chemical, physical and biological stability of food allergens and their allergenicity. They also suggest immunoblotting experiments using characterized IgE or IgG antibodies might also provide clues in further enhancing the understanding the allergenicity of food proteins.

References

1. Zhang, Y., et al. (2014) “Expression, Purification, and Characterization of Almond (Prunus dulcis) Allergen Pru du 4.”, J Agric Food Chem. 2014 Dec 31;62(52):12695-700. doi: 10.1021/jf5045102. Epub 2014 Dec 15.