title: Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma.
abstract: 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.
authors.affiliation: Unidad de Bioquimica, Departamento de Biotecnología, E.T.S. Ingenieros Agronomos, Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Palacin
authors.forename: J
authors.initials: J
authors.lastname: Varela
authors.forename: S
authors.initials: S
authors.lastname: Quirce
authors.forename: V
authors.initials: V
authors.lastname: del Pozo
authors.forename: L
authors.initials: L
authors.lastname: Tordesillas
authors.forename: P
authors.initials: P
authors.lastname: Barranco
authors.forename: M
authors.initials: M
authors.lastname: Fernandez-Nieto
authors.forename: J
authors.initials: J
authors.lastname: Sastre
authors.forename: A
authors.initials: A
authors.lastname: Diaz-Perales
authors.forename: G
authors.initials: G
authors.lastname: Salcedo
mesh_terms: D000328:Adult; D000485:Allergens / Q000097:blood / Q000737:chemistry / Q000276:immunology; D001249:Asthma / Q000175:diagnosis* / Q000276:immunology; D002352:Carrier Proteins / Q000097:blood / Q000737:chemistry* / Q000276:immunology; D002942:Circular Dichroism; D005260:Female; D005433:Flour; D005512:Food Hypersensitivity / Q000175:diagnosis* / Q000276:immunology; D006358:Hot Temperature; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology; D019570:Inhalation Exposure; D008297:Male; D008875:Middle Aged; D009784:Occupational Diseases / Q000175:diagnosis* / Q000276:immunology; D011489:Protein Denaturation; D055550:Protein Stability; D011994:Recombinant Proteins / Q000097:blood / Q000737:chemistry / Q000276:immunology; D014908:Triticum / Q000737:chemistry / Q000276:immunology*
chemical_list: D000485:Allergens; D002352:Carrier Proteins; D011994:Recombinant Proteins; C030363:lipid transfer protein; D007073:Immunoglobulin E