Cloning, Expression and Patient IgE Reactivity of Recombinant Pru du 6, an 11S Globulin from Almond

Abstract

Background: IgE-reactive proteins have been identified in almond; however, few have been cloned and tested for specific patient IgE reactivity. Here, we clone and express prunin 1 and prunin 2, isoforms of the major almond protein prunin, an 11S globulin, and assay each for IgE reactivity. Methods: Prunin isoforms were PCR-amplified from an almond cDNA library, sequenced, cloned and expressed in Escherichia coli. Reactivity to the recombinant (r) allergens, Pru du 6.01 and Pru du 6.02, was screened by dot blot and immunoblot assays using sera from almond-allergic patients and murine monoclonal antibodies (mAbs). Sequential IgE-binding epitopes were identified by solid-phase overlapping peptide analysis. Epitope stability was assessed by assaying denatured recombinant proteins by immunoblot. Results: IgE reactivity to rPru du 6.01 and rPru du 6.02 was found in 9 of 18 (50%) and 5 of 18 patients (28%), respectively. Four patients (22%) demonstrated reactivity to both isoforms. Murine anti-almond IgG mAbs also showed greater reactivity to rPru du 6.01 than to rPru du 6.02. Both stable and labile epitopes were detected. Six IgE-binding sequential epitope-bearing peptide segments on Pru du 6.01 and 8 on Pru du 6.02 were detected using pooled almond-allergic sera. Conclusions: rPru du 6.01 is more widely recognized than rPru du 6.02 in our patient population. The identification of multiple sequential epitopes and the observation that treatment with denaturing agents had little effect on IgE-binding intensity in some patients suggests an important role for sequential epitopes on prunins.

Scientific Opinion on the evaluation of allergenic foods and food ingredients for labelling purposes

“…Two isoforms have been identified: Pru du 6.01 and Pru du 6.02. Pru du 6.01 was recognised by 50 % and Pru du 6.02 by 28 % of almond-allergic patients (Willison et al, 2011).…”

Advances in allergen-microarray technology for diagnosis and monitoring of allergy: The MeDALL allergen-chip

“…Recombinant allergens were generated as previously described for Ana o 1 [19], Ana o 2 [20], Ana o 3 [21], Pru du 4 [22], Pru du 6 [23] and Pis v 3 [24].…”

“…Recombinant Pru du 3 was amplified directly from the almond cDNA library (library generation described in detail [23]). The sequence for the lipid transfer protein from almond (Prunus dulcis) was available in the NCBI database, accession number CAA65477.…”

Recombinant Allergen Production in E. coli

“…However, removal of the tag may result in nonspecific degradation or protein insolubilization. In such cases, the recombinant allergens can be analyzed without cleavage of the fusion protein [8]. 3.…”

“…MW: molecular weight standards; rPru du 6.01: recombinant Pru du 6.01-maltose binding protein (MBP) fusion protein; rPru du 6.02: recombinant Pru du 6.02-MBP fusion protein; nPru du 6: native amandin purified by column chromatography production of recombinant food allergens is available in references [5,6] and specific kit manuals. Protocols we used to produce recombinant allergens of almond [7,8], cashew [9][10][11], pecan [12,13], and pistachio [14] are described in this chapter. …”