New technologies for development of recombinant allergens
Project No. 01.2.2-LMT-K-718-01-0008
The project funded by Research Council of Lithuania for Targeted Research in Smart Specialisation Areas Executed by Vilnius University, Life sciences center, Institute of Biotechnology (2018-2022)
New universal platform for expression and purification of recombinant protein allergens have been applied for the selected human allergens in different hosts. The method is based on construction of allergen fusions with maltose binding protein (MBP) supporting the proper folding of the passenger protein and effective purification process using affinity chromatography. The obtained results show that the platform is well applicable for bacterial expression systems (more than 40 allergens tested). Yeast, plant and mammalian cell derived MBP fused with allergens are more sensitive to the host cell protein modifications different from the natural allergens, but still could be applicable in the cases, when bacterial hosts show incorrect alterations in expressed allergen structure. Demonstrating application opportunities of the produced and purified allergens, majority of them have provided the positive binding results with allergic human IgE antibodies responsible for the allergy diseases (ELISA and Western blot data).
New food allergen Cyp c 2 (beta enolase) was identified from the Lithuanian carp species and placed in Allergen Nomenclature data base, (WHO/IUIS Allergen Nomenclature Sub-Committee). The monoclonal antibodies specific to some allergens have been produced including Cyp c 1, Cyp c 2, Gad m 1, Der p 21, Der p 23. The conditions for obtaining recombinant allergen without MBP if necessary (after digestion with TEV protease) were selected as well as the conditions for storage and conservation of purified recombinant allergens. Constructed allergen expression plasmids (more than 30) have been deposited in Belgium collection of microorganisms and plasmids (see below: BCCM/GeneCorner) for open-access in R&D and allergy investigation.
This work was supported by the Europian Regional Development Fund (Project No. 01.2.2-LMT-K-718-01-0008) under grant agreement with Research Council of Lithuania (LMTLT).
Project leader dr. G. Žvirblis