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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.

Project leader dr. G. Žvirblis


Allergen constructions prepared and purified in the Project

Plasmid name* Insert coding allergen fused to MBP Origin of allergen
* Plasmid backbone is pET-28a-MBP-TEV: https://www.addgene.org/69929/
Plasmids are resistant to kanamycin; cultivation medium is LB-Lennox, cultivation temperature 25-37°C and IPTG induction;
Expression strains could be E.coli strains designed for pET plasmid, e.g.: BL21(DE3), Tuner(DE3), Rossetta (DE3), Origami™ 2 (DE3);
Plasmids are designed to express fused MBP-allergen protein containing TEV protease site for MBP removal if necessary;
Plasmid sequence data please find in BCCM/GeneCorner catalogue page.
** Der p 23 contains 6xHis tag at C-terminus.
*** Fel d 1 contains N-terminal chain-2 connected via linker (4Gly-Ser)3 to the chain-1.
pET-28a-MBP-TEV-Artv1 Art v 1 Artemisia vulgaris pollen
pET-28a-MBP-TEV-Betv1 Bet v 1 Betula pendula pollen
pET-28a-MBP-TEV-Canf5 Can f 5 Canis lupus familiaris kallikrein-related peptidase 2
pET-28a-MBP-TEV-Cypc1 Cyp c 1 Cyprinus carpio parvalbumin beta
pET-28a-MBP-TEV-Derf1 Der f 1 Dermatophagoides farinae mite
pET-28a-MBP-TEV-Derf2 Der f 2 Dermatophagoides farinae mite
pET-28a-MBP-TEV-Derp10 Der p 10 Dermatophagoides pteronyssinus tropomyosin
pET-28a-MBP-Derp21 Der p 21 Dermatophagoides pteronyssinus mite
pET-28a-MBP-TEV-Derp23 Der p 23** Dermatophagoides pteronyssinus endochitinase-like
pET-28a-MBP-TEV-Derp36 Der p 36 Dermatophagoides pteronyssinus mite
pET-28a-MBP-TEV-Feld1 Fel d 1 (2-1)*** Felis catus allergen, polypeptide chains 1 and 2
pET-28a-MBP-TEV-Feld4 Fel d 4 Felis catus allergen
pET-28a-MBP-TEV-Gadc1 Gad c 1 Gadus morhua callarias parvalbumin beta
pET-28a-MBP-TEV-Gald4 Gal d 4 Gallus gallus lysozyme
pET-28a-MBP-TEV-Gald8 Gal d 8 Gallus gallus parvalbumin alpha
pET-28a-MBP-TEV-Sals1 Sal s 1 Salmo salar parvalbumin beta
pET-28a-MBP-TEV-Cypc2 Cyp c 2 Cyprinus carpio enolase beta
pET-28a-MBP-TEV-Cypc3 Cyp c 3 Cyprinus carpio fructose-bisphosphate aldolase A
pET-28a-MBP-TEV-Cluh1 Clu h 1 Clupea harengus parvalbumin beta
pET-28a-MBP-TEV-Gadm1 Gad m 1 Gadus morhua parvalbumin beta
pET-28a-MBP-TEV-Eurm2 Eur m 2 Euroglyphus maynei mite
pET-28a-MBP-TEV-Derp26 Der p 26 Dermatophagoides pteronyssinus mite
pET-28a-MBP-TEV-Blot5 Blo t 5 Blomia tropicalis mite
pET-28a-MBP-TEV-Feld3 Fel d 3 Felis catus cystatin A
pET-28a-MBP-TEV-Phlp1 Phl p 1 Phleum pratense pollen
pET-28a-MBP-TEV-Canf8 Can f 8 Canis lupus familiaris cholesterol transporter 2
pET-28a-MBP-TEV-Shark Shark parvalbumin Triakis semifasciata (Leopard shark) parvalbumin alpha
pET-28a-MBP-TEV-Esolu1 Eso lu 1 Esox lucius (Northern pike) parvalbumin beta
pET-28a-MBP-TEV-Oncm1 Onc m 1 Oncorhynchus mykiss (Rainbow trout) parvalbumin beta
pET-28a-MBP-TEV-Scos1 Sco s 1 Scomber scombrus parvalbumin beta

Allergen expression plasmids available from BCCM/GeneCorner catalogue

No Plasmid name Accession No
1 pET-28a-MBP-TEV-Artv1 LMBP 11975
2 pET-28a-MBP-TEV-Betv1 LMBP 11976
3 pET-28a-MBP-TEV-Canf5 LMBP 11977
4 pET-28a-MBP-TEV-Cypc1 LMBP 11978
5 pET-28a-MBP-TEV-Derf1 LMBP 11979
6 pET-28a-MBP-TEV-Derf2 LMBP 11980
7 pET-28a-MBP-TEV-Derp10 LMBP 11981
8 pET-28a-MBP-TEV-Derp21 LMBP 11982
9 pET-28a-MBP-TEV-Derp23 LMBP 11983
10 pET-28a-MBP-TEV-Derp36 LMBP 11984
11 pET-28a-MBP-TEV-Feld1 LMBP 11985
12 pET-28a-MBP-TEV-Feld4 LMBP 11986
13 pET-28a-MBP-TEV-Gadc1 LMBP 11987
14 pET-28a-MBP-TEV-Gald4 LMBP 11988
15 pET-28a-MBP-TEV-Gald8 LMBP 11989
16 pET-28a-MBP-TEV-Sals1 LMBP 11990
17 pET-28a-MBP-TEV-Cypc2 LMBP 12818
18 pET-28a-MBP-TEV-Cypc3 LMBP 12819
19 pET-28a-MBP-TEV-Cluh1 LMBP 12820
20 pET-28a-MBP-TEV-Gadm1 LMBP 12821
21 pET-28a-MBP-TEV-Eurm2 LMBP 12822
22 pET-28a-MBP-TEV-Derp26 LMBP 12823
23 pET-28a-MBP-TEV-Blot5 LMBP 12824
24 pET-28a-MBP-TEV-Feld3 LMBP 12825
25 pET-28a-MBP-TEV-Phlp1 LMBP 12826
26 pET-28a-MBP-TEV-Canf8 LMBP 12827
27 pET-28a-MBP-TEV-Canf7 LMBP 12828
28 pET-28a-MBP-TEV-Shark LMBP 12829
29 pET-28a-MBP-TEV-Esolu1 LMBP 12830
30 pET-28a-MBP-TEV-Oncm1 LMBP 12831
31 pET-28a-MBP-TEV-Scos1 LMBP 12832
32 pET-28a-MBP-TEV-Sebm1 LMBP 12833
33 pET-28a-MBP-TEV-Lepw1 LMBP 12834