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Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma
Author(s) -
Palacin A.,
Varela J.,
Quirce S.,
Del Pozo V.,
Tordesillas L.,
Barranco P.,
FernandezNieto M.,
Sastre J.,
DiazPerales A.,
Salcedo G.
Publication year - 2009
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1111/j.1365-2222.2009.03280.x
Subject(s) - plant lipid transfer proteins , recombinant dna , asthma , proteolytic enzymes , chemistry , biochemistry , medicine , immunology , enzyme , gene
Summary Background Baker's asthma is an important occupational allergic disease. Wheat lipid transfer protein (LTP) Tri a 14 is a major allergen associated with wheat allergy. No panel of wheat recombinant allergens for component‐resolved diagnosis of baker's asthma is currently available. Objective To evaluate the potential role of recombinant Tri a 14 as a novel tool for the diagnosis of baker's asthma, and to test the heat and proteolytic resistance of the wheat LTP allergen. Methods A cDNA encoding Tri a 14 was isolated and sequenced, the recombinant allergen produced in Pichia pastoris and purified by chromatographic methods. Physicochemical and immunological comparison of the natural and recombinant forms of Tri a 14 was carried out by N‐terminal amino acid sequencing, matrix‐assisted laser desorption/ionization mass spectrometry, circular dichroism (CD) analysis, IgE immunodetection, and specific IgE determination and ELISA‐inhibition assays using a pool or individual sera from 26 patients with baker's asthma. Thermal denaturation and simulated gastrointestinal digestion of both Tri a 14 forms were checked by spectroscopic and electrophoretic methods, respectively, and biological activity by basophil activation test (BAT). Results Natural and recombinant Tri a 14 were similarly folded, as indicated by their nearly identical CD spectra and heat denaturation profiles. A high interclass correlation coefficient (0.882) was found between specific IgE levels to both Tri a 14 proteins in individual sera from baker's asthma patients, but a slightly lower IgE‐binding potency of rTri a 14 was detected by ELISA‐inhibition assays. Natural and recombinant Tri a 14 elicited positive BAT in two and one out of three patients, respectively. Heat denaturation profiles and simulated gastrointestinal digestion assays indicated that Tri a 14 displayed a high heat and digestive proteolytic resistance, comparable to those of peach Pru p 3, the model food allergen of the LTP family. Conclusions Recombinant Tri a 14 is a potential tool for baker's asthma diagnosis, based on its physicochemical and immunological similarity with its natural counterpart. Wheat Tri a 14 shows a high thermal stability and resistance to gastrointestinal digestion.