Engineering of the flavin-binding protein dodecin
We are currently evaluating Mycobacterium tuberculosis (mtDod) as a versatile platform for various applications, capitalizing on its ability to display peptides or proteins when fused to its N- or C-terminus. These fusion partners are presented on the outer surface of the dodecin cage, creating a well-defined and highly stable nanoscale scaffold. Owing to these properties, dodecin holds strong potential for immunization and vaccine development. Effective epitope presentation matrices should elicit high-quality antibody responses, while being cost-efficient to produce, stable without cold-chain requirements, and easy to deploy without medically trained personnel. mtDod fulfills all these criteria, making it a promising candidate for next-generation, accessible vaccine platforms.
Key structural properties:
The mtDod fold is found in prokaryotes only, and does not occur in eukaryotes.
It is a dodecameric protein; built up by tetramers of trimers.
C- and N-termini (12 x C, and 12 x N) are well exposed at the surface and accessible, and can be modified with peptides, proteins and other chemical moieties.
The mtDod scaffold is tolerant towards modification at its termini, allowing for example the attachment of the three times larger GFP 1.
The structure of mtDod and other dodecins have been reported at atomic resolution, such that structural features of the protein are known in detail.
Key functional properties:
mtDod shows high thermal stability and remarkable resistance towards chemicals); i.e., it is stable to up to several hundred mM hydrochloric acid.
Heterododecameric complexes of two mtDod constructs can be formed at different composition (by polycistronic expression of mtDod variants or refolding from mixtures of variants), which depend on the relative concentration of constructs available during protein assembly.
Heterododecameric complexes enable the packing of dodecin in heterovalent fashion which imposes advantages in immunizations.
mtDod is generally sufficiently large to provoke an immune response in mammals (shown for rabbits) and avians (shown for chicken). For a pilot study on the production of diagnostic antibodies from rabbits, see Bourdeaux et al. (ref 2). Further, as has been shown by others, mtDod can improve immunogenicity and protective efficiency of epitopes in vaccine development against tuberculosis 4.
Protein production:
mtDod and mtDod-peptide fusions are accessible from recombinant production in E. coli in yields of up to one gram per liter shake flask batch culture.
The proteins are generally received in pure form via protocols based on heat-denaturation and precipitation (chromatography not required).
mtDod has also been synthesized by microwave assisted solid phase peptide synthesis (company CEM, USA) and refolded to oligomeric protein.
Other production hosts (e.g., baker’s yeast) or cell-free-expression systems seem suitable.
Current areas of research aim at harnessing mtDod as carrier in immune-engineering; i.e., as carrier for peptides by expressing dodecin-peptide fusions.
1. Grininger, M., Staudt, H., Johansson, P., Wachtveitl, J. & Oesterhelt, D. Dodecin is the key player in flavin homeostasis of Archaea. J Biol Chem 284, 13068–13076 (2009).
2. Ludwig, P. et al. Characterization of the small flavin-binding dodecin in the roseoflavin producer Streptomyces davawensis. Microbiology 164, 908–919 (2018).
3. Bourdeaux, F. et al. Comparative biochemical and structural analysis of the flavin-binding dodecins from Streptomyces davaonensis and Streptomyces coelicolor reveals striking differences with regard to multimerization. Microbiology 165, 1095–1106 (2019).
4. Bourdeaux, F. et al. Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin. ACS Infect. Dis. 4, 1082–1092 (2018).
5. Bourdeaux, F. et al. Dodecin as carrier protein for immunizations and bioengineering applications. Sci. Rep. 10, 13297 (2020).