Dodecin as carrier protein for immunizations and bioengineering applications

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).

For being suited as scaffolds, proteins need to meet an array of requirements. Depending on the actual use, multiple features of the protein can be important; e.g., particle size, achievable purity, expression level, robustness of fold/assembly, general stability and immunogenicity (if used for immunizations). Two key features are obligatory, in addition. Scaffolds need to form a stable and water-soluble structure that is best insensitive to the attached cargo, and they should further allow the dense packing of the cargo in homovalent and ideally also in heterovalent fashion [1][2][3][4][5] . One application of scaffold proteins is their conjugation with peptides for the generation of antibodies (AB), utilizing the increased immunogenicity of the protein-peptide conjugate (in this role often called carrier proteins) 6 . Such ABs can be used to identify proteins, which contain the peptides used for AB generation, in complex samples, and allow the specific labelling of proteins of interest in their spatiotemporal distribution, e.g. by immunofluorescence imaging or western blotting. For the reactivity of the ABs, the selection of the peptide is critical, since the ABs derived from the conjugate can only recognize the peptide as presented (or similar) on the carrier 7 . For the recognition of the protein in its native form, the correct sequence, but also the structure and surface exposure of the selected peptide need to be considered 7 . For B-cell activation, the conjugated peptide needs to be exposed on the surface of the carrier, and it is thought that a dense packing of the conjugated peptide is advantageous for this, because highly repetitive epitopes on the particle/carrier surface facilitate B-cell receptor oligomerization 1,2 .
Usually peptide-carrier conjugates for AB production are formed by coupling an about 20 amino acid-long peptide to residues at the surface of a carrier protein via a chemical reaction [8][9][10] . Commonly used carrier proteins are keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and rabbit serum albumin (RSA), but also other proteins, e.g. tetanus toxoid (TT), and artificial carrier-systems, e.g. multiple antigen peptides (MAP) or virus-like particles (VLP, not limited to chemical conjugations), are used 11,12 . While BSA bears typical carrier properties (likely also other albumins), and exposes the peptides at the surface at a potentially high density 13,14 , KLH is often preferred as a carrier-protein due to its high immunogenicity 15,16 . Notably, the immune system reacts to the entire conjugate, and, therefore, ABs are not just raised against the peptide of interest, but also against the carrier protein and the linker (peptide or remnant of the coupling agent/crosslinker). To avoid cross-reactivity by anti-carrier or anti-linker ABs, it is beneficial to use carrier-linker systems for immunization that have no or only very low similarity with the inventory of cells and tissues that are supposed to be analysed 16 .
Although the method of chemically coupling peptides of interest to carrier proteins is commonly used, it is not without weaknesses. Besides limitations arising from solid support synthesis of the peptide themselves, e.g. limited solubility of hydrophobic sequences or amyloid forming sequences, the spectrum of peptides that can be coupled to the above listed carrier proteins is constrained by its compatible with the coupling agent. For example, internal cysteine residues are avoided, because they are commonly terminally introduced for the coupling to the carrier protein [8][9][10] . Further, in some cases, the stability of the conjugation product or intermediate (activated carrier) can be problematic 10,17 . An alternative method to the coupling approach is the direct expression of self-assembling peptide-carrier conjugates, provided as encoding DNA sequence to the recombinant expression host. This approach allows more flexibility in the design of epitopes and linkers, since the limitations of coupling reactions or peptide synthesis need not be taken into account. Self-assembling carrier proteins can also be produced with tags or proteins that allow post-translational covalent linking of cargo, thereby not relying Scientific RepoRtS | (2020) 10:13297 | https://doi.org/10.1038/s41598-020-69990-0 www.nature.com/scientificreports/ on chemical coupling 18,19 . While this carrier method has high potential, it is reliant on the availability of stable and robust self-assembling protein-and peptide-scaffolds. The dodecin protein family was recently discovered as a flavin storage and buffering system that occurs in bacteria and archaea, but not in eukaryotes [20][21][22][23] . Dodecins are 8 kDa small proteins of βαββ-topology. Although forming a small antiparallel β-sheet that partly enwraps the helix, the dodecin fold is unique. Dodecins largely meet the requirements of protein scaffolds. In the native dodecameric state, dodecins are of spherical shape with 23-cubic symmetry, and the N-and C-termini are exposed at the protein surface. Dodecins show pronounced thermostability (> 95 °C) [22][23][24] , which likely originates from an extensive antiparallel β-sheet that is built upon protomer assembly.
Here, we present dodecin from Mycobacterium tuberculosis (mtDod) as a new carrier protein for peptides and scaffold for bioengineering applications. To evaluate mtDod's suitability as a carrier/scaffold protein, we expressed and purified diverse mtDod fusion constructs, and analysed the obtained dodecamers. The robustness of the dodecamer assembly was probed by the ability to express the diverse constructs as dodecamers in Escherichia coli. Further, we analysed the stability of the obtained dodecamers, and found that it is largely unaffected by the attached tag, linker and/or cargo. Finally, we demonstrate that the use of mtDod as a carrier benefits from its accessibility in high amounts via by a simple heat denaturation protocol. MtDod conjugates with heat sensitive cargo were purified via conventional affinity chromatography.
The exposed termini of mtDod can be harnessed for charging/functionalization in diverse ways of which we used two in this study: First, a cargo was directly fused by attaching the peptide/protein-encoding sequence at the gene level. Second, mtDod was terminally modified with conjugation sites that allow post-translational covalent and non-covalent fusions of the peptide/protein as well as other chemical entities to the intact dodecin carrier (Fig. 1). In a case study, we directly tested the suitability of mtDod as carrier for producing anti-peptide ABs for laboratory use. ABs were raised in rabbits against mtDod-peptide fusions, and showed comparable labelling capability as commercially available ABs judged by western blotting.

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. All constructs were expressed in E. coli BL21 (DE3). Cells were grown in terrific broth (TB) medium to an optical density at 600 nm (OD 600 ) of about  www.nature.com/scientificreports/ 0.6-0.8 at 37 °C before induction with isopropyl-β-D-thiogalactopyranoside (IPTG; 0.5 mM final concentration), and expression was performed over night at 20 °C. Since mtDod is a flavin binding protein (preferred flavin-ligand is riboflavin-5′-phosphate (FMN)) 23,24 , its overexpression causes increased amounts of cellular flavin, leading to a yellowish colouring of the cells. Cells were lysed by French press, and the cell debris was removed by centrifugation. Depending on the mtDod construct different purification strategies were applied. In the following, construct names are underlying the nomenclature: peptides or proteins fused to the N-terminus of a protein are written in front of the protein (e.g. peptide-mtDod) and C-terminal fusions are written after the protein (e.g. mtDod-peptide). Most mtDod constructs were produced as soluble proteins, but some proteins, such as mtDod-SZ1, mtDod-SZ3 (SYNZIP constructs) 28 , H8-SpyC-mtDod and mtDod-SpyC-H8 (SpyC constructs) 25,26 accumulated as inclusion bodies (Table 1). For soluble mtDod constructs, most cytosolic E. coli proteins were removed by heat denaturation at about 75 °C. MtDod itself is stable to temperatures above 95 °C under standard conditions (pH ~ 7.5 and ionic strength > 100 mM, e.g., in PBS), and the thermal stability can be further increased by adding the native FMN ligand in excess 23,24 . Depending on the stability of the fused cargo, lower temperatures during the heat denaturation may be necessary, or different purification approaches need to be applied (affinity chromatography). For example, mtDod-mmACP started to precipitate at about 55-60 °C in spite of mtDod staying intact, as indicated by maintained FMN binding and preserved dodecameric stability ( Supplementary Fig. S1). In this case, heat denaturation was conducted at about 55 °C. Lower temperatures during the heat denaturation step can affect the purity of preparations, because some E. coli proteins remain soluble. Following heat treatment, mtDod constructs were generally further purified by two cycles of DMSO-induced precipitation (50% final DMSO concentration). Finally, size-exclusion chromatography (SEC) was performed to select for dodecameric fractions, which can be Table 1. Selection of mtDod constructs used for expression studies are divided into two groups: mtDodpeptides (constructs with only short peptides fused to mtDod) and mtDod-proteins (constructs with domains or entire proteins fused to mtDod). mtDod(WT): wild type mtDod. seACP: Saccharopolyspora erythraea ACP, gene chlB2. msfGFP: monomeric superfolder green fluorescent protein 29 . For a full description of constructs, see Supplementary Table S1. The constructs mtDod-GSG-Lys and mtDod-PAS-Met have been used as control; MtDod-GSG-Lys was used for its flexible linker, and mtDod-PAS-Met for its rigid linker. The PAS linker were based on sequences (slight alterations) shown in ref 30 . # Describes whether the major fraction of the expressed construct is soluble or forms inclusion bodies. MtDod constructs with low solubility form often non-classical inclusion bodies (correctly folded protein) 31 , leading to their yellowish colouring (flavin binding). Since flavin binding only requires intact mtDod, it is possible that inclusion bodies are yellow although the protein cargo is misfolded. *MtDod-SpyC-H8 seems to be soluble in cellular environment but forms yellow aggregates after cell lysis. **SZ1-mtDod and SZ3-mtDod also formed inclusion bodies and behaved similarly as the C-terminal constructs (data not shown). ***MtDod-seACP could not be obtained in soluble form; under all applied refolding conditions yellow aggregate was formed.  Fig. S2). For larger mtDod constructs with fused proteins, like mtDod-mmACP (Fig. 2a), significant aggregation was observed in the SEC profiles (see Supplementary Fig. S2). For the construct mtDod-msfGFP-H8, purification by heat denaturation (70 °C, above that aggregation was observed) and purification by affinity chromatography were compared. GFP is a suitable cargo for this test, because GFP is highly thermostable 32 . The dodecameric structure of dodecin causes a high density of surface exposed affinity tags, allowing vigorous washing without severe protein loss during Ni-chelating affinity chromatography. Accordingly, mtDod-msfGFP-H8 was washed with two column volumes of a 200 mM imidazolecontaining wash buffer, and elution was performed at 400 mM imidazole. While with both purification strategies mtDod-msfGFP-H8 dodecamer was obtained, the sample purified by heat denaturation showed severe aggregation in SEC ( Supplementary Fig. S3).
MtDod constructs that aggregate in inclusion bodies can be refolded by dialysis, as previously described 23 , under conditions optimized for the respective fused cargo. All inclusion bodies were first washed and then dissolved by denaturation using 6 M guanidinium chloride. MtDod was refolded without further purification at different conditions ranging from pH 5.0 23 to pH 8.5. Refolding was possible for all constructs obtained as inclusion bodies in this study, although the resolubilized proteins remained aggregation-prone, particularly during protein concentration and filtration. For a screen of buffer conditions for refolding constructs mtDod-SpyC-H8 and H8-SpyC-mtDod, see Supplementary Fig. S4. Notably, for both constructs, a glycerol-containing buffer was found to be best suited for refolding.
Overall, all constructs presented in Table 1, except mtDod-seACP, were obtained in high purity (see Fig. 2b). We thought that the insolubility and aggregation problems observed for some constructs may be solved by the formation of mtDod-heterododecamers, because then the density of entities on the surface could be reduced. To probe heterododecamer formation with mtDod in vitro and in vivo, we worked with the two species mtDod-PAS-Strep and mtDod(WT). We note that mtDod-PAS-Strep was used for its availability in the lab and is not compromised in solubility. We assume that other mtDod-PAS-peptide constructs than mtDod-PAS-Strep could have been used, too. For in vitro heterododecamer formation, mtDod(WT) and mtDod-PAS-Strep were jointly refolded in different relative concentrations, while for the formation of heterododecamers in vivo, After the heat treatment, the pH was increased to about 6.8 using a glycerol-and Tris-HCl-containing buffer, followed by a second heat treatment (5 min 95 °C). MtDod-SZ1 and mtDod-SZ2 were denatured with loading buffer containing ~ 7 M urea and 2.5% SDS (prolonged heat treatment: 15 min 95 °C). Denaturation with acidic loading buffer was more reliable for most constructs and easier in handling compared to urea-based protocols (in some cases even 7-8 M urea failed to dissociate the protein completely). Of note, when treated with acidic loading buffer, some constructs showed additional bands (mainly SpyC constructs, Supplementary Fig. S5). The origin of this behaviour was not further investigated.  30 . Data indicates that the composition of heterododecamers is controlled by the relative concentration of species in the refolding solution,i.e., the higher the concentration of a construct, the more abundant it is in the refolded dodecamer. Of note, assuming that the heterododecamer formation is just controlled by the concentration of each construct (see Fig. 3a), the band patterns for heterododecamers assembled in vivo can be used to estimate gene order related expression strength (see Fig. 3b), as described in the literature for other methods, e.g. FRET 33 . The estimated relative expression strength of each gene is for the bicistronic vector: first > second, and for the tricistronic vector first > third > second.

Scientific RepoRtS
Dodecin is highly stable. We have recently established the cyclic thermal shift assay, termed thermocyclic fluorescence assay, to determine the stability of dodecins 23 . This assay is based on the fluorescence quenching that is observed when flavins bind to dodecin. In each binding pocket of the dodecamer, the two isoalloxazine ring systems of two bound flavins are embedded between symmetry-related tryptophans. 22−34 Since dodecins can only bind flavins in the dodecameric state, the fluorescence intensity of flavins can be used to estimate the amount of dodecameric mtDod in solution. In contrast to standard melting analysis, in which the temperature is continuously increased, the thermocyclic fluorescence assay runs cyclic temperature profiles that contain a heating phase (temperature increased per cycle) and a cooling phase (for all cycles cooled to 5 °C). At the heating phase, FMN is released from the binding pocket and the fluorescence intensity increases. During cooling, FMN can rebind to the dodecamer (cooling phase) restoring initial low fluorescence values. As soon as the dodecamer denatures irreversibly, the fluorescence intensity remains at elevated levels. By plotting the fluorescence intensity of the cooling phase against the heating phase temperature, the thermal stability of the dodecamer of the mtDod constructs can be determined. Since all constructs, except mtDod-SZ1 and mtDod-SpyC-H8, proved to be stable in PBS buffer throughout the entire temperature range, we identified the slightly destabilizing conditions of pH 4.2 as suited to sense the impact of the cargo on the integrity of the mtDod dodecameric scaffold (see Fig. 4). Under this condition, the thermally stable constructs mtDod(WT) and mtDod-peptides started to denature at 75-80 °C. Of note, we considered a protein to denature when the fluorescence curve reached its knee right before going into a steep increase in fluorescence reaching values of above 30%. It is further important to note that the thermocyclic fluorescence assay does only monitor the dodecameric stability, which may be influenced by the attached cargo. For The band at about 40 kDa represents likely the dodecamer and the band above seems to be caused by the mild denaturation conditions during sample preparation. The appearance of additional bands at higher molecular weight is also observable in other lanes. We want to note that these bands don't depict the hexamer and the dodecamer, as all proteinprotein interactions, which would stabilize the hexamer, are present in higher numbers in the dodecamer with also other additional stabilizing interactions. The origin of the mtDod dodecamer migration behaviour and the molecular mechanism behind the bands at higher molecular weight is not clear. www.nature.com/scientificreports/ 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 also 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. In accordance to the lower stability at pH 4.2, observed in the thermocyclic fluorescence assay, a two-component acidic loading buffer (3.3% SDS and pH < 4.2 during heat treatment, afterwards 2.5% SDS and pH 6.8) was applied to fully denature the dodecamer (Fig. 2).
While we did not study the effects of freezing and thawing explicitly, we would like to note that we did not observe noticeable aggregation for most mtDod constructs after freezing and thawing (all constructs presented here were frozen and thawed at least once). However, constructs that are prone to aggregation might be problematic during freezing and thawing. Accordingly, we noticed aggregation for mtDod-msfGFP-H8, indicated by green fluorescent aggregates after thawing, and mtDod SYNZIP constructs, forming yellowish precipitate. For SpyC mtDod constructs, glycerol containing buffer could prevent noticeable aggregation after freezing and thawing. peptides/proteins fused to mtDod remain functional. The accessibility and functionality of folds and peptides fused to mtDod were tested by the reactivity of the SpyT/-C and SnpT/-C pairs [25][26][27] . These systems allow the covalent conjugation between two entities of which one is equipped with a peptide tag (Tag) and the other with a small protein fold (Catcher) 26 . Applications range from attaching proteins from pathogens to scaffolds, like VLPs and IMX313 (heptamer forming coiled coils), for immunizations 18,19 , to recruiting enzymes to a scaffold hub for creating assemblies with elevated substrate turnover 35 . In this study, seACP-SpyC and mClover3-SnpC were prepared as cargo for performing SpyT/-C and SnpT/-C reactions with the respective Tag-labelled mtDod constructs. For the inverse reaction, mtDod SpyC constructs and SpyT-seACP were used. For all reactions, the scaffold was saturated with two molar equivalents of cargo. The reactions were incubated for 20 h at 22 °C, and analysed by SDS-PAGE (Fig. 5).
For all combinations of mtDod scaffold and cargo, the expected product band(s) of mtDod and the specific cargo(s) were observed in SDS-PAGE. While for mtDod SpyT/SnpT constructs no unreacted scaffold proteins was observed, for the inverse setting, with mtDod SpyC constructs, bands of unreacted scaffold monomer were visible (possibly caused by aggregation problems of the mtDod SpyC constructs). We note that mtDod SpyT/ www.nature.com/scientificreports/ SnpT constructs are lower in molecular mass than the mtDod SpyC constructs, and traces of unreacted scaffold protein may be less visible on SDS-PAGE gels. This data shows that a high degree of saturation was achieved, indicating that SpyT/-C and SnpT/-C are well accessible at the mtDod dodecamer scaffold. Double-tagged constructs SpyT-mtDod-SnpT or SnpT-mtDod-SpyT, heterovalently loaded with seACP-SpyC and mClover3-SnpC, revealed bands of single-charged mtDod monomers in SDS-PAGE. We explain this observation by an increased density at the surface of mtDod that sterically constrains the conjugation with both cargos. Similar as the SpyT/-C and SnpT/-C constructs, also the SYNZIP constructs can be used for recruiting proteins to the mtDod scaffold (although non-covalently). Due to the limited solubility and high aggregation tendencies of SYNZIP constructs, we only tested if mtDod SYNZIP constructs are able to interact with the respective SYNZIP counterpart (e.g., mtDod-SZ1 with SZ2-mClover3). For both mtDod SYNZIP constructs, we observed the formation of mtDod cargo adducts, indicated by higher apparent molecular mass peaks in SEC ( Supplementary Fig. S8). This shows that also SYNZIP domains fused to mtDod are functional and accessible. However, we deemed the SpyT/-C and SnpT/-C systems more suitable for mtDod constructs, and did not further investigated the SYNZIP system. In order to probe the accessibility and functionality of linked folds further, we tested the labelling of mmACP linked to mtDod with a 4′-phosphopantetheine CoA fluorophore mediated by the 4′-phosphopantetheine transferase from Bacillus subtilis (Sfp). The Sfp-mediated modification of ACP with CoA-modified fluorophores (CoA-488; ATTO-TEC dye ATTO 488) has been frequently used for the labelling of cellular compounds 36 . All reactions were conducted at 25 °C for 1 h in triplicates, and stopped by the addition of acidic loading buffer and analysed by SDS-PAGE. To determine the relative accessibility of mmACP linked to mtDod, fluorescence intensities of mtDod-mmACP and mtDod-mmACP-H8 were compared to free mmACP after labelling (Fig. 6).
By comparing the fluorescence intensities of CoA-488-labeled mtDod-mmACP and mtDod-mmACP-H8 with CoA-488-labeled free mmACP, the relative degree of labelling was determined to about 31% ± 8% and 36% ± 8% respectively. After an additional hour of labelling, in-gel fluorescence of mtDod-mmACP and of mtDod-mmACP-H8 further increased by 14% ± 8% and 24% ± 12%, respectively. The overall low relative degree of labelling and the increase after an additional hour of reaction time indicates a reduced accessibility of mmACP fused to mtDod. It cannot be ruled out that the mmACP fold fused to mtDod is instable or partly unfolded. Note that in SDS-PAGE, mtDod-mmACP runs at just two different apparent molecular weights corresponding to labelled and non-labelled protein (see Fig. 6). It seems that SDS-PAGE is limited in its efficiency of separating mixtures of unlabelled and labelled monomers.
MtDod-PAS-pep constructs for AB production. Protein carriers are generally used for the production of ABs against peptides or proteins 9 . In the standard approach, the peptide or the protein of interest is linked to the carrier, usually BSA or KLH, by chemical ligation [8][9][10] . While the method is well-established and broadly used for AB production, problems can arise during conjugating the peptide/hapten to the carrier, e.g., owing to the low stability or solubility of the conjugate (or even for the peptide alone) or altered antigenic properties of the peptide 17 . The dodecameric structure with the exposed termini allows mtDod to be charged with 12 or 24 peptides/proteins on its surface by simply fusing the peptide/protein encoding sequence to the mtDod gene. In order to evaluate the suitability of mtDod for AB production, 11 fusion constructs were produced in E. coli of which each is comprised of mtDod, a PAS linker and a peptide of interest, termed mtDod-PAS-Pep (Table 2). Peptide sequences originated from human heat shock proteins (HSP), proheparin-binding EGF-like growth factor (HB-EGF) and C-terminus of the heat shock cognate protein 70 interacting protein (CHIP) (for detailed peptide origin see Supplementary Table S2). Peptides/epitopes were selected, because of a specific scientific interest in In all reactions, bands of higher mass representing the conjugation products are observed. As mentioned above, the acidic loading buffer causes the appearance of double bands (mtDod SpyC constructs) and smearing bands (seACP-SpyC-H8) for some constructs.
Scientific RepoRtS | (2020) 10:13297 | https://doi.org/10.1038/s41598-020-69990-0 www.nature.com/scientificreports/ the proteins carrying the peptides, and not for their sequence composition or the thermal stability properties of the source proteins (e.g. thermal stability of HSP). In that light, the case study presented here is also a "real case" for the applicability of the dodecin matrix. Pep-encoding sequences, provided on oligonucleotide primers, were introduced in single-step by ligationfree cloning. Recombinant expressions and purifications followed the established protocols described above. All constructs were received as soluble proteins, except mtDod-PAS-Pep7 that formed inclusion bodies (see Table 2).  www.nature.com/scientificreports/ The yellow colour of the inclusion bodies indicated assembled dodecamer, and we assume that aggregation of mtDod-PAS-Pep7 was induced by the cysteine in Pep7, forming disulfide-bridges between the dodecamers. All constructs, except mtDod-PAS-Pep7, were further purified by two cycles of DMSO-induced precipitations. FMN was added before constructs were eventually forwarded to SEC to remove unbound FMN and remaining DMSO as well as to select for dodecameric species (Fig. 7a). We assumed that mtDod remains saturated with FMN due to its high affinity and cooperative binding mode. 23 FMN-saturated mtDod constructs (FMN:mtDod constructs) can be determined in concentration by absorbance at 450 nm, and are amenable to stability measurements by the thermocyclic fluorescence assay. All constructs were received as dodecamers, as indicated by SEC ( Supplementary  Fig. S10). MtDod-PAS-Pep3 shows in addition to the dodecamer species higher oligomeric states in SEC, which we assume to result from disulfide-bridges formed by the cysteine in Pep3. The dodecamer containing fractions were pooled, and the purity was controlled by SDS-PAGE (see Fig. 7b). The thermocyclic fluorescence assay revealed the high thermal stability of all mtDod-PAS-Pep constructs, similar as the wild type protein (Fig. 7c) 23 . Molecular masses of the constructs were measured with ESI-MS and confirmed full-length protein (see Table 2).  Purified mtDod constructs were eventually submitted to an AB production company for immunization in rabbits and AB purification (Davids Biotechnologie GmbH, Germany). AB productions were induced in one rabbit for each construct by 5 injections (about 100 µg each, mtDod-PAS-Pep solution concentration: 2.2-7.5 mg/mL (average 4.5 ± 1.3 mg)) over 63 days using the adjuvant MF59/AddaVax. The ABs were purified from the collected serum by affinity chromatography with the respective mtDod-PAS-Pep construct immobilized on the column matrix. For all the 10 mtDod-PAS-Pep constructs, which were submitted to immunizations, purified ABs were obtained and their binding behaviour analysed by western blotting (summarized in Table 3).
The produced ABs did not create any reoccurring background signals indicating that the mtDod-PAS carrier matrix does not cause the generation of ABs that recognize proteins present in lysate of HEK293T cells (see Fig. 8a, Supplementary Fig. S11 and Supplementary Fig. S12). This agrees with the low sequence identity of mtDod-PAS with human proteins (protein-protein BLAST with default settings finds no human protein with significant similarity).
The six ABs rated as "class 1" were tested on different concentrations of target protein (1 µg to 60 ng) to estimate their general labelling capability in western blots (see Fig. 8b). Further, the same dilutions of target protein were used to give a rough comparison for mtDod-PAS-Pep derived ABs with commercially available ABs (exception mtDod-PAS-Pep2, for which we had no commercial AB to hand). In general, the mtDod-PAS-Pep derived ABs were comparable with commercial ABs (see Fig. 8b), and are well suited for specific labelling of target protein in lysate samples.
The mtDod-PAS-Pep-derived ABs show that mtDod-PAS is a well-suited carrier system for the production of peptide-specific ABs. MtDod-PAS-based fusions benefit from the easy cloning, uncomplicated production/ purification and the high protein yields. In our case study, problems in the purification of cysteine containing constructs emerged from the oxidative conditions induced by high FMN concentrations, which may, however, be overcome when working under reducing conditions. Alternatively, cysteine could be replaced by serine residues, as recommended by AB producing companies for epitopes containing internal cysteines, although this might alter the epitopes and affect the specificity of the produced ABs. Table 3. Classification of mtDod-PAS-Pep derived ABs. ABs were classified as followed: "Class 1" ABs recognize the proteins of interest provided as recombinantly purified protein (produced in E. coli). "Class 2" ABs do not recognize recombinant protein of interest, but protein of expected apparent molecular weight in HEK293T human cell lysates. "Class 3" ABs only recognize mtDod-PAS constructs (like the respective mtDod-PAS-Pep or mtDod-PAS-Met). Western blots of ABs rated as "class 1" or "class 2" are shown in Fig. 8. The AB derived from mtDod-PAS-Pep6 and mtDod-PAS-Pep8 only recognized mtDod-PAS-Pep constructs and were considered "class 3" (Supplementary Fig. S11). *mtDod-PAS-Pep5 derived ABs showed only a very weak signal for 1 µg and 500 ng of recombinant protein with no intensity difference (see Supplementary Fig. S11). Thus, the AB preparation was regarded as "class 2". **Proheparin-binding EGF-like growth factor of Chlorocebus aethiops (green monkey). ***mtDod-PAS-Pep10 derived ABs didn't recognize purified CHIP but seem to recognize a protein in CHIP-overexpressing cells; no detection range was determined.

mtDod-PAS-Pep4
HSP-A4 C-terminus Class 1 www.nature.com/scientificreports/ www.nature.com/scientificreports/ conclusion During the last years, dodecins have been characterized as flavin binding proteins involved in flavin homeostasis 21,23 . In addition, the unique protein fold and particularly the exceptional flavin binding mode were harnessed in technological applications, although exclusively on the archaeal protein from Halobacterium salinarum 41,42 . In this study, we present mtDod as a versatile scaffold protein to attach peptides and small proteins. The mtDod dodecamer tolerates high temperatures and various chemical conditions, which allows protein purification by quick heat-induced denaturation and protein precipitation with solvents, and holds out the prospect that mtDod broadly accept conditions for chemical ligation reactions. In addition to its high stability, mtDod can be produced in high amounts in E. coli. For soluble constructs, yields of up to several hundred milligrams of mtDod-peptide fusions per litre of bacterial culture can be expected. Proteins fused to mtDod are presented at the mtDod outer surface, and have been shown to remain accessible and functional. Both the SpyT/-C and the ACP/Sfp system allowed attaching the cargo at the mtDod surface. In this respect, mtDod is comparable to the recently presented IMX313 scaffold, suggested for use in vaccine development 19 . When evaluating mtDod as a scaffold, we observed that constructs can suffer from low solubility in response to the properties of the attached cargo. While agglomeration by disulfide formation, observed for mtDod-PAS-Pep3 and mtDod-PAS-Pep7, could simply be avoided by reducing conditions during protein preparation or replacing of cysteine with serine residues, solubility problems induced by hydrophobic and structurally unstable fold may be solved by heterododecamer formation to dilute the aggregation-inducing species on the mtDod surface. For the construct mtDod-PAS-Strep, we demonstrated that heterododecamer formation with the wild type protein is readily possible in vitro and in vivo by simply providing both proteins during refolding or recombinant protein production, respectively (see Fig. 3).

mtDod-PAS-Pep5
As a pilot run for evaluating the suitability of mtDod as a carrier matrix for AB production, we chose 11 peptides originating from different human proteins like CHIP or HSP-70, and fused them to mtDod. One of the 11 peptide constructs formed inclusion bodies, while all other constructs were purified by the standard heat-denaturation purification protocol without any need for individual optimization. From all immunizations performed in this study, ABs were received that at least recognized the mtDod-PAS scaffold in western blotting. Overall, 8 of the 10 ABs recognized proteins in HEK293T human cell lysate at expected molecular weight. For 6 of them, correct target recognition could be confirmed with the recombinantly purified protein as reference. No AB preparation showed any unspecific reactivity in HEK293T cell lysate, demonstrating that mtDod is a suitable matrix for the production of ABs that specifically label the proteins of interest (in the HEK293T lysate) without the need to remove anti-carrier ABs. The low sequence identity of mtDod to human proteins and eukaryotic proteins in general suggests that ABs derived from mtDod will also be specific to proteins of interest in other human samples/cells 20,24 .
The here presented AB production strategy with mtDod may be attractive for labs that are experienced in protein expression, and want to produce ABs targeting peptides without relying on peptide synthesis and chemical crosslinking. We expect that the exposed termini are also suited for chemical ligation of haptens or antigens, following standard immunization protocols, or for Click chemical modification 43 . However, in proofing the concept of dodecin for peptide immunizations, we did not elaborate on this further. Finally, we note that the availability of dodecins with similar features (e.g. Streptomyces coelicolor, Streptomyces davaonensis and Thermus thermophilus dodecins) 22,44 is advantageous when aiming for heterologous prime/boost protocols by using two dodecin scaffolds with low sequence identity fused with the same antigen 45 .
While the mtDod has been mainly tested as carrier matrix for AB production in this study, the properties of mtDod call for its application as a scaffold in a broad range of biotechnological and bioengineering applications. We encourage to explore the mtDod as a scaffold when defined particles with specific surface properties are required. Such constructs can be valuable in e.g. diffusion measurements, 46 for formation of biomaterials [47][48][49][50] and in creating enzyme scaffolds 35,51,52 . MtDod heterododecamers may be applied for pull down assays when combining a mtDod construct bearing a protein recruiting peptide and a mtDod construct with a purification tag.

Material and methods
cloning. Expression constructs were cloned using standard PCR methods and In-Fusion HD Cloning (TaKaRa Bio Europe). Primers were ordered from Sigma-Aldrich. Inserts were verified by Sanger sequencing (by Microsynth Seqlab, Göttingen, Germany). For polycistronic constructs spacer DNA sequences (between genes) were designed with EGNAS (version 1,158) 53 . For a list of all constructs see Supplementary Table S1. expression and cell lysis. Plasmids were transformed into BL21 (DE3) Gold cells and cells were plated onto LB-agar plates containing 100 ng/µL ampicillin and 1 g/mL glucose. 10 mL LB medium with 100 ng/µL ampicillin and 1 g/mL glucose were inoculated with a single colony and incubated at 37 °C and 180 rpm overnight. 1 L TB medium with 100 ng/µL ampicillin was inoculated with 10 mL overnight LB culture, and incubated at 37 °C and 180 rpm until the OD 600 reached about 0.8. The cultures were cooled to 20-30 °C, and the expression was induced with 1 mL 1 M IPTG solution. The cultures were incubated overnight at 20 °C 160 rpm for protein production. MtDod-PAS-Pep constructs were expressed in 500 mL TB medium induced with 500 µL 1 M IPTG. Cells were harvested at 4,000 rcf and frozen in liquid nitrogen or directly processed. For purification by heat denaturation (mtDod constructs), cell pellets were resuspended in 30 mL standard dodecin buffer: 300 mM NaCl, 5 mM MgCl 2 and 20 mM Tris-HCl (pH 7.4, adjusted with HCl). For purification by His-tag affinity chromatography, cell pellets were resuspended in 30 mL Ni-NTA wash buffer I: 200 mM NaCl, 35 mM K 2 HPO 4 and 15 mM KH 2 PO 4 (pH 7.4, adjusted with NaOH or HCl) and 40 mM imidazole. To the resuspended cells, PMSF and DNase I were added, and cells were disrupted by French press. Cell debris was removed by centrifugation (50,000 rcf, 20 min). All steps after cell harvest were conducted at 4 °C or on ice. ). Plates were placed on ice and 2 µL of the corresponding 50 mM mtDod construct solution were added to the wells. Plates were then sealed with optical tape (iCycler iQ; Bio-Rad Laboratories, Inc.), centrifuged (3,000 rcf, 2 min) and placed into a precooled (5 °C) real-time PCR instrument (C1000 Thermal Cycler and CFX96 Real-Time System; Bio-Rad Laboratories, Inc.). For the fluorescence detection, excitation/emission filter bandwidth of 450-490/560-580 nm was used. After 1 h incubation at 5 °C, the heating and cooling cycles were started, with each cycle containing a heating phase for 6 min and a cooling (5 °C) phase for 30 min. The heating phase temperature was raised stepwise from 5 °C to 95 °C. Until 50 °C, the step size was 4.5 °C while at higher temperatures the step size was reduced to 2.0 °C. Data points were taken after each phase. The complete temperature protocol was applied to every sample. For the stability measurements of mtDod-PAS-Pep constructs, the acetate buffer was replaced with a MES buffer (150 mM NaCl, 100 mM MES (pH 4.7, adjusted with HCl) and the heating phase duration was prolonged to 10 min. Spyc and Snpc reactions. The reactions were carried out in phosphate borate buffer (pH 8.5) at 25 °C for 20 h. Concentration of the respective carrier constructs were 10 µM (1 eq.) and 20 µM of the respective cargo constructs (2 eq.), reaction volume was 50 µL. Each protein also was separately prepared in the same concentration as used in the reaction and incubated under the same conditions. 5 µL of each reaction and control were analysed by SDS-PAGE and Coomassie staining, for all samples the acidic loading buffer was used.

Lc-MS.
For the LC-MS analysis of the mtDod-PAS-Pep constructs, 500 µg protein were precipitated with 75% (v/v) acetone (− 20 °C, final concentration), pelleted by centrifugation (20,000 rcf, 5 min) and dissolved in 100 µL water. After removal of undissolved aggregates by centrifugation (20,000 rcf, 5 min), the solution was diluted with 5% (v/v) acetonitrile:water to a final concentration of 0.1 mg/mL. The injection/sample size was 2.5 µL (250 ng). Samples were analysed by using a Dionex UltiMate 3,000 RSLC (Thermo Fischer Scientific) coupled to a micrOTOF-Q II (Bruker Daltonik GmbH) equipped with an electrospray ionization source. Chromatographic separation (further desalting) was performed on a Discovery BIO Wide Pore C5 column (100 × 2.1 mm, particle size 3 μm, Sigma-Aldrich) at 55 °C with a mobile-phase system consisting of water and acetonitrile (each containing 0.1% formic acid). A linear gradient ranging from 5 to 95% acetonitrile over 14 min at a flow rate of 0.4 mL min −1 was used. MS data was acquired in positive mode in the range from 200-2,500 m/z and later analysed using Compass DataAnalysis 4.0 software (Bruker Daltonik GmbH).