Selective Cloning of Peanut Allergens, Including Profilin and 2S Albumins, by Phage Display Technology

Abstract

Background: Peanut kernels contain many allergens able to elicit IgE–mediated type 1 allergic reactions in sensitized individuals. Sera from sensitized patients recognize variable patterns of IgE–binding proteins. The identification of the IgE–binding proteins of peanut extract would faciliate improvement of diagnostic and immunotherapeutic approaches as well as development of sensitive test systems for the detection of hidden peanut allergens present as additives in various industrial food products and the investigation of their stability during processing of food products. Methods: We applied the pJuFo cloning system based on the phage surface display of functional cDNA expression products to clone cDNAs encoding peanut allergens. Sera (n = 40) of peanut–allergic individuals were selected according to case history, radioallergosorbent test and immunoblot analysis to demonstrate IgE binding towards the newly identified recombinant allergens. Results: In addition to the known allergens Ara h 1 and Ara h 2 we were able to identify four allergens with estimated molecular weights of 36, 16, 14.5 and 14 kDa. Three of them formally termed Ara h 4, Ara h6 and Ara h 7 show significant sequence similarities to the family of seed storage proteins and the fourth (Ara h 5) corresponds to the well–known plant allergen profilin. Immunoblotting of the six expressed recombinant allergens with 40 patients sera shows 14 individual recognition patterns and the following frequency of specific IgE binding: Ara h 1 was recognized by 65%, Ara h 2 by 85%, Ara h 4 by 53%, Ara h 5 by 13%, Ara h 6 by 38% and Ara h 7 by 43% of the selected sera. Conclusions: All of the selected peanut–positive sera can detect at least one of the six identified recombinant allergens which can be used to establish individual patients’ reactivity profiles. A comparison of these profiles with the clinical data will possibly allow a further insight into the relationship between clinical severity of the symptoms and specific IgE levels towards the six peanut allergens.

Temporal and Spatial Expression of the Major Allergens in Developing and Germinating Peanut Seed

“…Although about 11 peanut allergens have been reported (www.allergome.org), Ara h 1, Ara h 2, and Ara h 3 are classified as the major peanut allergens because they are generally recognized by more than 50% of peanut-allergic patients (Koppelman et al, 2001). Specifically, Ara h 1 and Ara h 2 are recognized by 70% to 90% of patients with peanut allergy (Burks et al, 1995bClarke et al, 1998), and Ara h 3 is recognized by serum IgE from approximately 44% to 54% of different patient populations with a history of peanut sensitivity (Kleber-Janke et al, 1999;Rabjohn et al, 1999).…”

Cloning and structural analysis of a cDNA clone encoding glycinin (Gly-1) seed storage protein of peanut

“…Other two genes are -Ara h3 isolated at University of Arkansas (Rabjohn et al 1999) and Ara h4 isolated from Borstel, Germany (Kleber-Janke et al 1999) has shown IgE mediated allergic reaction (Kleber-Janke et al 1999;Rabjohn et al 1999). A detailed understanding of the protein structure and diversity is an important prerequisite for attempts to manipulate quality because it indicates the extent to which the structure of the proteins can be manipulated without affecting their biological properties (Shewry and Casey, 1999).…”

“…The mature proteins are an oligomer of six similar (hexameric) subunits (Badley et al 1975;Shewry et al 1995). Peanut contains multiple seed storage proteins some of the arachins (glycinins) are identified as allergens, which are responsible for triggering IgE mediated allergic reaction (Rabjohn et al 1999;Kleber-Janke et al 1999;Viquez et al 2003;Koppleman et al 2004). High sequence homology of peanut Gly-1 with Ara h3 and Ara h4 suggest that peanut Gly-1 also carry corresponding four IgE binding regions (R1 consists of amino acid residue 42 to 77; R2 consists of amino acid residue 157 to 177; R3 consists of amino acid residue 250 to 288 and R4 consists of amino acid residue 290 to 347 of the peanut Gly-1 protein).…”

“…Earlier studies with peanut proteins have demonstrated that peanut glycinin is composed of multiple subunits (Krishna and Mitra, 1987). Two other glycinin genes Ara h3 (Rabjohn et al 1999) and Ara h4 (Kleber-Janke et al 1999) from peanut have been isolated and characterized for their allergic property. In legumes, the 11S seed storage (legumins/glycinins) genes are the major storage proteins, presence of multiple subunits for these proteins is a common feature (Argos et al 1985;Shewry et al 1995) and an individual gene encodes for one subunit (Nielsen et al 1989).…”

Identification of Borrelia burgdorferi Ribosomal Protein L25 by the Phage Surface Display Method and Evaluation of the Protein's Value for Serodiagnosis

“…This technology has previously been applied successfully to identify allergens from cDNA libraries of Aspergillus fumigatus (5), Cladosporium herbarum (29), peanuts (12), and mites (8) by employing IgE antibodies from patients. Genomic phage surface display libraries of three different Borrelia strains and one mixed library consisting of all three strains were constructed by helper phage superinfection according to a published protocol (2).…”