Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy

Kan Takanobu

Hiragun Takaaki

Ishii Kaori

Hiragun Makiko

Yanase Yuhki

Tanaka Akio

Hide Michihiro

Allergology International

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 timeconsuming. 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 b-hexosaminidase release tests with RBL-48 cells. PrMGL_ 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.

HRT

MGL_1304

Pichia pastoris

Sweat allergy

β-hexosaminidase release

Medical sciences [ 490 ]

eng

Japanese Society of Allergology

Copyright © 2015, Japanese Society of Allergology. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/)

[ISSN] 1323-8930

[DOI] 10.1016/j.alit.2015.03.003

[PMID] 26117259

[DOI] https://doi.org/10.1016/j.alit.2015.03.003