Dodecin as carrier protein for immunizations and bioengineering applications

Florian Bourdeaux,Martin Grininger,Hüseyin Besir,Ines Gößner,Yannick Kopp,Julia Lautenschläger,R Martin Vabulas

doi:10.1038/s41598-020-69990-0

Abstract

In bioengineering, scaffold proteins have been increasingly used to recruit molecules to parts of a cell, or to enhance the efficacy of biosynthetic or signalling pathways. For example, scaffolds can be used to make weak or non-immunogenic small molecules immunogenic by attaching them to the scaffold, in this role called carrier. Here, we present the dodecin from Mycobacterium tuberculosis (mtDod) as a new scaffold protein. MtDod is a homododecameric complex of spherical shape, high stability and robust assembly, which allows the attachment of cargo at its surface. We show that mtDod, either directly loaded with cargo or equipped with domains for non-covalent and covalent loading of cargo, can be produced recombinantly in high quantity and quality in Escherichia coli. Fusions of mtDod with proteins of up to four times the size of mtDod, e.g. with monomeric superfolder green fluorescent protein creating a 437 kDa large dodecamer, were successfully purified, showing mtDod’s ability to function as recruitment hub. Further, mtDod equipped with SYNZIP and SpyCatcher domains for post-translational recruitment of cargo was prepared of which the mtDod/SpyCatcher system proved to be particularly useful. In a case study, we finally show that mtDod-peptide fusions allow producing antibodies against human heat shock proteins and the C-terminus of heat shock cognate 70 interacting protein (CHIP).

Highlights

LAL Limulus amebocyte lysate Lys Lysate multiple antigen peptides (MAP) Multiple antigen peptides mmACP Mus musculus acyl carrier protein msfGFP Monomeric superfolder green fluorescent protein mtDod Mycobacterium tuberculosis dodecin mtDod(WT) Mycobacterium tuberculosis dodecin wild type OD600 Optical density at 600 nm OE Over expressing cells rabbit serum albumin (RSA) Rabbit serum albumin SCBT Santa Cruz Biotechnology seACP Saccharopolyspora erythraea acyl carrier protein size-exclusion chromatography (SEC) Size exclusion chromatography Sfp 4′-Phosphopantetheine transferase from Bacillus subtilis Sigma Sigma-Aldrich SnpC SnoopCatcher SnpT SnoopTag SpyC SpyCatcher SpyT SpyTag SZ SYNZIP domain Tag Small peptide sequence that interacts with Catcher’s (SpyTag or SnoopTag) terrific broth (TB) Terrific broth TBS Tris-HCl buffered saline TBST Tris-HCl buffered saline with Tween-20 tetanus toxoid (TT) Tetanus toxoid VLP Virus-like particle
Dodecins have been characterized as flavin binding proteins involved in flavin homeostasis[21,23]
The unique protein fold and the exceptional flavin binding mode were harnessed in technological applications, exclusively on the archaeal protein from Halobacterium salinarum[41,42]

Summary

Results and discussion

Dodecin can be recombinantly produced in high yields. To evaluate the suitability of mtDod as a carrier protein, several mtDod constructs were designed and purified. For. example, in screening temperatures for the heat denaturation of mtDod-mmACP, we observed the formation of yellowish agglomerates above 55–60 °C, indicating that the construct is intact in the mtDod scaffold, as capable of FMN binding, but precipitated by the thermally unfolded mmACP (band representing the intact dodecamer observable in SDS PAGE, see Supplementary Fig. S7). The high dodecameric stability of mtDod is observed in SDS-PAGE using the standard loading buffer (2.5% SDS, pH 6.8) (see Supplementary Fig. S5) Under these conditions, further depending on the heat treatment for sample preparation, a dodecameric fraction remains intact, as indicated by the high molecular weight band representing the dodecamer. MtDod-PAS-Pep[3] and Dod-PAS-Pep[6] contained

C-terminus

Conclusion

Material and methods

Methods