title: Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy.
abstract: BACKGROUND
  We previously identified MGL_1304 secreted by Malassezia globosa as a sweat antigen for patients with atopic dermatitis (AD) and cholinergic urticaria (ChU). However, purifying native MGL_1304 from human sweat or culture supernatant of M. globosa (sup-MGL_1304) is costly and time-consuming. Moreover, recombinant MGL_1304 expressed by using Escherichia coli (TF-rMGL_1304) needs a large chaperon protein and lacks the original glycosylation of yeasts. Thus, we generated a recombinant MGL_1304 by Pichia pastoris (P-rMGL_1304) and investigated its characteristic features.
  
  
  METHODS
  Recombinant MGL_1304 proteins expressed by E. coli and P. pastoris were generated. Properties of these recombinants and native antigens were compared by western blot analysis, histamine release tests (HRT) of patients with AD and ChU, and β-hexosaminidase release tests with RBL-48 cells. P-rMGL_1304-specific IgE in sera of patients with AD were measured by sandwich ELISA.
  
  
  RESULTS
  Western blot analysis revealed that IgE of patients with AD bound to all MGL_1304 recombinants and native antigens. The histamine releasing ability of P-rMGL_1304 was 100 times higher than that of TF-rMGL_1304, and was comparable to that of sup-MGL_1304. Degranulation rates of RBL-48 cells, sensitized with sera of patients with AD in response to the stimulation of P-rMGL_1304, were comparable to those of sup-MGL_1304, whereas those of TF-rMGL_1304 were relatively weak. The levels of P-rMGL_1304-specific IgE in sera of patients with AD were correlated with their disease severities.
  
  
  CONCLUSIONS
  P-rMGL_1304 has an antigenicity comparable to the native antigen, and is more useful than TF-rMGL_1304, especially in HRT and degranulation assay of RBL-48 cells.
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Takanobu
authors.initials: T
authors.lastname: Kan
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Hiragun
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Ishii
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Makiko
authors.initials: M
authors.lastname: Hiragun
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Yuhki
authors.initials: Y
authors.lastname: Yanase
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Department of Dermatology, Integrated Health Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. Electronic address: ed1h-w1de-road@hiroshima-u.ac.jp.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Hide
mesh_terms: D000485:Allergens / Q000276:immunology; D000818:Animals; D015549:Basophil Degranulation Test; D002460:Cell Line; D003876:Dermatitis, Atopic / Q000175:diagnosis* / Q000276:immunology; D003881:Dermatomycoses / Q000175:diagnosis* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D005656:Fungal Proteins / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000175:diagnosis* / Q000276:immunology; D008290:Malassezia / Q000276:immunology*; D010843:Pichia / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000276:immunology; D013542:Sweat / Q000276:immunology; D014581:Urticaria / Q000175:diagnosis* / Q000276:immunology
chemical_list: D000485:Allergens; D005656:Fungal Proteins; D011994:Recombinant Proteins
keywords: HRT; MGL_1304; Pichia pastoris; Sweat allergy; β-hexosaminidase release