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Purification and characterisation of the dimeric group 12 allergen from Blomia tropicalis heterologously expressed by Escherichia coli Top10F´

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Abstract

Successful research in the wide-ranging field of allergy is usually achieved by definition not only of physicochemical and immunological properties of natural, but also recombinant allergens. Blomia tropicalis mite is a well-known source for various groups of hypersensitivity-causing proteins. The goal of the present work was to produce, purify and characterise by in silico, biochemical and immunological methods the recombinant group-12 allergen of B. tropicalis. The recombinant Blo t 12 aggregation capacity as well as the affinity to antibodies from BALB/c immunised mice and B. tropicalis-sensitised human donors were investigated through in silico analyses, dynamic light scattering, SDS-PAGE, ELISA and Western blot. The presence of Blo t 12 within B. tropicalis extracts was also determined by ELISA and Western blot. High concentrations of dimeric rBlo t 12 were detected through SDS-PAGE next to other aggregates and the results were confirmed by data from DLS and Western blot. The YITVM peptide was predicted to be the most aggregation-prone region. The IgE-reactivity of rBlo t 12 was not completely abolished by aggregate formation but it was significantly decreased compared to rBlo t 5, or B. tropicalis extracts. Natural Blo t 12 may naturally dimerises, but it was detected in non-delipidified B. tropicalis extracts in low amounts. Given that this allergen may be a specific marker for B. tropicalis allergy, the recombinant Blo t 12 herein obtained is characterised as a mid-tier allergen in Brazilian atopic patients and may be useful for the improvement in precision allergy molecular diagnostic applications.

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Acknowledgements

ESS was the recipient of a scholarship from the Research's Foundation of the State of Bahia (FAPESB grant BOL0449/2015). LGCP and NMAN are recipients of research fellowships from National Council for Scientific and Technological Development of Brazil (CNPq).

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Coordination for the Improvement of Higher Education Personnel (CAPES), Project No. 077/2012.

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Authors and Affiliations

  1. Department of Biointeraction, Federal University of Bahia, Salvador, Brazil

    Eduardo Santos da Silva, Luis Gustavo Carvalho Pacheco, Antônio Márcio Santana Fernandes, Elisânia Fontes Silveira, Carina da Silva Pinheiro & Neuza Maria Alcantara-Neves

  2. Department of Molecular and Cellular Anatomy, University of Regensburg, Regensburg, Germany

    Claudia Asam

  3. Laboratório de Alergia e Acarologia, Instituto de Ciências da Saúde, Universidade Federal da Bahia, Avenida Reitor Miguel Calmon, sem nº, Canela, CEP – 40110-100, Salvador, Bahia, Brazil

    Neuza Maria Alcantara-Neves

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Contributions

NM Alcantara-Neves designed the study and supervised the project. ES Silva significantly contributed to perform the present study, wrote the manuscript and conducted the in vitro and in vivo laboratory assays, as well as in silico analyses. CS Pinheiro co-supervised the project, helped with data analyses and manuscript revision. LGC Pacheco was involved in conception the project, revision/validation of in silico analysis assays and manuscript revision. AMS Fernandes performed IgE reactivity assay and helped with other experiments. C. Asam performed expression and purification pilot assays and manuscript revision. E.F. Silveira helped with laboratory assays, particularly DLS analyses. NM Alcantara-Neves and CS Pinheiro also drafted the manuscript. All authors read and approved the final manuscript.

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Correspondence to Neuza Maria Alcantara-Neves.

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The authors have no conflicts of interest.

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Ethics Committee on Research of the Faculty of Medicine of the Federal University of Bahia (CAAE 45376814.0.0000.5577) approved human sera use. Ethical Committee for Use of Experimental Animals of the Institute of Health Sciences of Federal University of Bahia (CEUA-ICS 137/2018) allowed mice experiments

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11033_2021_6361_MOESM1_ESM.tif

Supplementary file1 — Fig. S1 Heterologous expressions and solubility test. Expression of rBlo t 12 in 5 mL of LB medium using BL21(DE3) (a), BL21(DE3)pLys S and TOP 10 F’ strains (b). c Western blot for confirmation of expression, using anti-HIS IgG antibodies. The sample added is indicated above each lane. MW: molecular weight. d 18% SDS-PAGE for the solubility test. e Western blot analysis of the same samples shown in d. Above each lane is indicated the sampleadded in the wells. Arrows indicate the suggestive band for rBlo t 12.(TIF 10854 kb)

11033_2021_6361_MOESM2_ESM.tif

Supplementary file2 — Fig. S2 PASTA 2.0 and AGGRESCAN3D analyses [19,20]. a Energy scores of the aggregation pairings retrieved from PASTA 2.0 server. b Aggregation free energy profile. c Aggregation helix profile from PASTA 2.0. d Ribbon representation of the aggregation-prone region, with the penultimate coil and β-sheet of Blo t 12. e Modelled 3D 10 structure retrieved from AGGRESCAN3D, focusing in the prone-aggregating region YITVM and other A3D scores residues (all in red). (TIF 7804 kb)

11033_2021_6361_MOESM3_ESM.tif

Supplementary file3 — Fig. S3 Correlations of sIgE binding assays. a Spearman’s correlation of BtE ELISA results and sIgE from ImmunoCAP assay (n=30). b Spearman’s correlation of rBlo t 5 ELISA results and sIgE from ImmunoCAP assay (n=25). (TIF 4361kb)

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da Silva, E.S., Pacheco, L.G.C., Fernandes, A.M.S. et al. Purification and characterisation of the dimeric group 12 allergen from Blomia tropicalis heterologously expressed by Escherichia coli Top10F´. Mol Biol Rep 48, 3405–3416 (2021). https://doi.org/10.1007/s11033-021-06361-6

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