CARRIER MATRIX COMPRISING DODECIN PROTEIN

Abstract

The present invention relates to a carrier conjugate comprising at least one dodecin protein unit conjugated with at least one hapten and/or immunogenic and/or enzymatically active moiety. Further, the invention relates to a method for producing said conjugate and a method for producing antibodies that specifically binds to a hapten and/or immunogenic moiety of the conjugate, and to a method for performing enzymatic or diagnostic assays in vitro using said conjugate. Moreover, the invention relates to the use of said conjugate for producing antibodies that specifically bind to the epitope or epitopes contained in the moiety of said conjugate and use of said conjugate for performing enzymatic or diagnostic assays in vitro.

Claims

1. A conjugate comprising at least one dodecin protein unit conjugated with at least one hapten and/or at least one immunogenic and/or at least one enzymatically active moiety.

2. The conjugate according to claim 1, wherein said at least one hapten and/or at least one immunogenic and/or at least one enzymatically active moiety is complexed with and/or genetically fused to said dodecin protein unit.

3. The conjugate according to claim 1, wherein said dodecin protein is a dodecamer of twelve units of said dodecin protein, wherein said dodecamer comprises at least one conjugated dodecin protein unit conjugated with said at least one hapten and/or at least one immunogenic and/or said at least one enzymatically active moiety.

4. The conjugate according to claim 1, wherein said moiety is selected from an antigen, a peptide, an enzyme, a protein, a lipid, and a small molecule.

5. The conjugate according to claim 1, wherein said conjugation is covalent or non-covalent via a functional group, wherein said functional group is selected from a thiol group, an amino group, a carboxy group and an azide group.

6. The conjugate according to claim 1, wherein said fusion is direct or indirect via a suitable linker group or sequence.

7. The conjugate according to claim 1, wherein said dodecin protein unit conjugated with said at least one hapten and/or at least one immunogenic and/or at least one enzymatically active moiety is suitably labelled.

8. The conjugate according to claim 1, wherein said dodecin protein unit is derived from a bacterium or archaea selected from Halobacterium salinarum, Halobacterium halobium, Streptomyces davaonensis, Streptomyces coelicolor, Chlorobium tepedium, Sinorhizobium meliloti, Bordetella pertussis, Bordetella bronchiseptica, Pseudomonas aeruginosa, Pseudomonas putida, Acinetobacter baumannii, Thermus thermophilus, Geobacter sulfurreducens, and Mycobacterium tuberculosis.

9. A composition comprising the conjugate according to claim 1 and at least one of a suitable carrier, excipient, enzyme substrate or adjuvant.

10. A nucleic acid encoding a conjugate according to claim 1, or an expression vector expressing or overexpressing said nucleic acid.

11. A recombinant host cell, comprising the nucleic acid or the vector according to claim 10.

12. A method for producing a conjugate according to claim 1, the method comprising suitably culturing a recombinant host cell comprising and expressing a nucleic acid encoding a conjugate according to claim 1, or an expression vector expressing or overexpressing said nucleic acid, and isolating said conjugate from the cell and/or the culture medium thereof.

13. The method for producing a conjugate according to claim 1, the method comprising suitably culturing a recombinant host cell comprising and expressing a nucleic acid or a vector encoding a dodecin protein unit, isolating said dodecin protein unit; and complexing an hapten and/or immunogenic and/or enzymatically active moiety via a functional group to said isolated dodecin protein unit.

14. A method for producing an antibody that specifically binds to the hapten of the conjugate according to claim 1, comprises the steps of suitably immunizing a subject with the conjugate according to claim 1, and isolating an antibody specifically binding to the hapten of said conjugate from the subject, wherein said subject is an animal.

15. A method for performing an enzymatic assay in vitro, wherein said method comprises the steps of immobilizing at least one conjugate comprising an enzymatically active moiety according to claim 1 on a solid carrier; adding a suitable enzyme substrate; and determining the amount of converted enzyme substrate.

16. The conjugate according to claim 3, wherein both termini of the amino acid chain of said dodecin protein unit are located on the outer surface of said dodecamer.

17. The conjugate according to claim 6, wherein the fusion is via a peptide linker.

18. The method according to claim 15, wherein said solid carrier is selected from glass, agarose, polymers, and metal.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0075] FIG. 1 shows a schematic depiction of mtDod constructs. The constructs of the present invention can, for example, be produced in E. coli (left) and purified by a heat treatment protocol (middle). The dodecamers preferably expose their termini at the outer surface. These termini can then be attached to/loaded with peptides, proteins and small molecules (“cargo”) by attaching said cargo indirectly at “docking sites” (DD) ((i)-(iii)) or by directly fusing said cargo on the gene level (iv).

[0076] FIG. 2 shows an SDS PAGE gel of purified dodecin constructs. For full denaturing of the dodecamer an acidic loading buffer (pH 4.2) containing 3.3% SDS was used during the heat treatment (10 min, 95° C.). After the heat treatment, the pH was increased to about 6.8 using a glycerol and Tris-HCl containing buffer. For mtDod-msfGFP-H8 impurities of lower mass are observable which may be caused by mtDod with degraded msfGFP. The double bands observed for both SpyCatcher mtDod constructs seem to be caused by the acidic loading dye in combination with the SpyCatcher, as also seACP-SpyC formed double bands when the acidic loading dye was used. The origin of this behavior was not further investigated.

[0077] FIG. 3 shows the thermal stability of mtDod constructs determined with a termed thermocyclic fluorescence assay. The FMN fluorescence at the rebinding/cooling phase is plotted against the heating phase temperature. Increasing FMN fluorescence indicates disassembly of the dodecamer at the heating phase as the flavin cannot be rebound in the cooling phase and its fluorescence is not quenched. In PBS, no significant increase of fluorescence is observed, except for mtDod-SpyC and mtDod-SZ1, indicating that the dodecameric mtDod core structures of all other constructs does not disassemble. The minor increase of fluorescence of up to 20% around 45-50° C., might be caused by hindered rebinding of FMN and not by disassembling of the dodecamer. At pH 4.2, all constructs show a significant increase of fluorescence indicating the disassembly of the dodecamer. Most constructs behave like mtDod(WT) and are stable to about 80° C., except mtDod-PAS-Met, mtDod-mACP, mtDod-SpyC and SpyC-mtDod, of which the last three start denaturating already at 50° C. mtDod-PAS-Met is only slightly less stable and starts to denature around 75° C. Data for mtDod-SpyC and mtDod-SZ1 need careful evaluation, because both constructs have a significantly impaired FMN binding and were not saturated with FMN. The fluorescence was normalized with the maximal fluorescence measured in the heating phase corrected by the temperature-induced fluorescence decline of FMN.

[0078] FIG. 4 shows a SDS PAGE of the SpyTag/-Catcher and SnoopTag/-Catcher reactions. Left: Reactions of mtDod Spy-/SnoopTag constructs with seACP-SpyCatcher and/or mClover3-SnoopCatcher. Right: Inverse reactions of mtDod SpyCatcher constructs with SpyT-seACP. In all reactions, bands of higher mass representing the reaction product are observed. For both mtDod-SpyCatcher constructs and seACP-SpyCatcher double bands are observed, caused by the acidic loading dye.

[0079] FIG. 5 shows modification of mtDod-mACP and mACP by Sfp with fluorescent CoA. a) Coomassie stained SDS PAGE gel of the reaction solution and negative controls. Upper bands (slightly above the 25 kDa ladder band) represent SFP, middle bands (slightly above the 20 kDa ladder band) mtDod-mACP and lower bands (below 15 kDa ladder band) mACP. b) Fluorescence image of the SDS PAGE gel of the reaction solutions. Only proteins modified with the ATTO dye 488 are visible as a fluorescence band. Higher bands represent mtDod-mACP and lower bands mACP.

[0080] FIG. 6 shows MtDod-PAS-Pep purity and stability. a) SEC profiles of FMN:mtDod-PAS-Pep1. As only the dodecamer can bind flavins, the dodecamer peak can be identified by the absorption at 375 nm and 450 nm. In addition to the dodecamer, unbound FMN is visible at 120 mL. Except mtDod-PAS-Pep3, which formed higher oligomers in addition to the dodecamer, all constructs behaved similarly. b) SDS-PAGE gel of all purified mtDod-PAS-Pep constructs. In addition to the weak monomer band, also the dodecamer band is visible in the gel. Standard loading dye was used (30 min 95° C.; heat treatment) to obtain bands of the monomer and dodecamer. c) Thermocylic fluorescence assay of mtDod-PAS-Pep constructs.

[0081] FIG. 7 shows Western Blots with selected mtDod-PAS-Pep construct antibodies. a) Overall recognition of the target protein in purified form and in lysate (L or OE-L for lysate of over expressing cells). Antibodies did not show any cross-interference, but e.g. antibodies of mtDod-PAS-Pep2 recognize HSP70 to some the degree. Antibodies derived from mtDod-PAS-Pep3 required prolonged detection times (40 s (right) compared to 13 s (left)) to detect target protein in the lysate. Antibodies derived from mtDod-PAS-Pep10 did not recognize purified target protein (CHIP), but seem to recognize something with similar mass in CHIP overexpressing cells. b) Western Blots for comparison with commercially available antibodies. Different amounts (1 μg to 60 ng) of purified target protein (here HSP70 and HSP110) were loaded and then analyzed by mtDod-PAS-Pep derived antibodies (1 μg/mL) and commercial available antibodies (manufacturer's recommended concentration).

[0082] FIG. 8 shows confocal immunofluorescence images of mouse testis sections. The membrane of elongated spermatids recognized by the respective anti-S1c36a3 antibodies is displayed in light gray. DNA was coloured by Hoechst fluorescent dye and is displayed in dark gray. In this embodiment of the present invention, two epitopes A and B from the protein Slc36a3 derived from sperm of adult mice that were coupled to dodecin as a carrier are used. The construct produces antibodies that exhibit less background compared to a standard carrier-matrix KLH. Tested was a mixture of KLH-epitope(A)+KLH-epitope(B) (KLH_mix-A+B; same mixture used in two rabbits, 324 and 325) for antibody production in rabbits vs a mixture of mtDod-epitope(A)+mtDod-epitope(B) (mtDod Mix-A+B), and dodecin as modified on both termini epitope(A)−mtDod-epitope(B), mtDod Dual). Abbreviations: SAO359: Antibodies derived from a mixture of both mtDod epitope fusion constructs. SAO360: Antibodies derived from an mtDod epitope fusion construct with one epitope fused to the N-terminus and the other epitope fused to the C-terminus (see example 5).

EXAMPLES

[0083] Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the description, figures and tables set out herein. Such examples of the methods, uses and other aspects of the present invention are representative only, and should not be taken to limit the scope of the present invention to only such representative examples.

[0084] The following examples have been performed using dodecin from Mycobacterium tuberculosis (mtDod), but the person of skill will be able to readily apply and thus to transfer the teachings as herein to the inventive use of any other suitable dodecin protein.

Example 1

[0085] To evaluate the suitability of dodecin from Mycobacterium tuberculosis (mtDod) as a carrier protein, several mtDod constructs were designed and purified. All constructs were expressed in E. coli BL21 under the control of the lac promoter. Cells were grown in terrific broth (TB) medium to an OD of about 0.6-0.8 at 37° C. before induction with isopropyl-β-D-thiogalactopyranosid (IPTG; 0.5 mM final concentration), and expression was performed overnight at 20° C. Cells were lysed by French press, and the cell debris was removed by centrifugation. Most mtDod constructs were produced as soluble proteins, but some constructs were received in inclusion bodies.

[0086] For soluble mtDod constructs, a heat denaturation step at about 75° C. was performed in order to remove most cytosolic E. coli proteins. MtDod itself is stable to temperatures above 95° C. under standard buffer conditions (pH˜7.5 and ionic strength >100 mM, e.g., PBS) and the thermal stability can be further increased by native flavin mononucleotide (FMN) ligand added in excess [4, 5]. Depending on the stability of the fold of the cargo fused to mtDod, lower temperatures may be necessary during the heat denaturation step. For example, mtDod-mACP precipitates at about 55-60° C. In this case, heat denaturation was conducted at about 55° C. Lower temperatures during the heat denaturation step can lower the protein purities as some E. coli proteins stay in solution. In this case, purification by affinity chromatography, in order to circumvent heat treatment, may be a more suitable method.

[0087] After the removal of heat denatured and precipitated proteins by centrifugation, mtDod was generally further purified by two DMSO precipitations (50% final concentration). Here, mtDod precipitates, but can easily be dissolved in buffer again. Finally, size-exclusion chromatography (SEC) was performed to select for dodecameric fractions, identified in the chromatographic profiles by the bound flavin (absorption at 375 nm and 450 nm).

[0088] The two purification strategies; i.e., heat denaturation and affinity chromatography, were compared using the example of the construct mtDod-msfGFP-H8. GFP is a suited cargo for this test, because the high thermal stability of GFP allows heat denaturing at temperatures as used for mtDod (WT) [6]. The dodecameric structure of dodecin constructs causes a high density of affinity tags exposed at the surface allowing vigorously washing steps without severe protein loss during Ni-chelating affinity chromatography. MtDod-msfGFP-H8 was washed with 2 column volumes of a 200 mM imidazole containing wash buffer, while elution was performed at 400 mM imidazole.

[0089] Constructs that express in inclusion bodies can be refolded by dialysis as previously described in Bourdeaux et al. [4], with the optimal conditions depending on the 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 [4] to pH 8.5. In the context of the present examples for all constructs obtained as inclusion bodies, refolding was possible although the soluble proteins suffering from aggregation, particularly during protein concentration and filtration. For both SpyCatcher mtDod constructs a glycerol containing buffer was used.

[0090] Table 1 shows MtDod constructs that were used for expression studies. Most mtDod constructs expressed in soluble form, but some constructs formed yellowish inclusion bodies indicating partly correct folded dodecin. MtDod constructs are divided into two groups: mtDod-peptides (constructs with only short peptides fused to mtDod) and mtDod-proteins (constructs with whole proteinaceous domains or proteins fused to mtDod).

TABLE-US-00001 Molar mass/ Construct Linker system Da Expression state mtDod-peptides mtDod(WT) —  7497.41 soluble mtDod-GSG-Lys GSG  8411.37 soluble mtDod-PAS-Met PAS  8875.93 soluble mtDod-SpyT PASG 10457.72 soluble SpyT-mtDod GPAS 10215.45 soluble mtDod-PAS2-SpyT PAS2G 11446.78 soluble SpyT-PAS2-mtDod GPAS2 11204.52 soluble SpyT-mtDod-SnpT GPAS/PASG 13141.70 soluble mtDod-proteins mtDod-mACP PAS 19333.60 soluble mtDod-msfGFP-H8 PAS 36387.68 soluble mtDod-SpyC-H8* PAS 22412.60 inclusion body H8-SpyC-mtDod PAS 22072.19 inclusion body mtDod-SZ1 PAS 14231.99 inclusion body SZ3-mtDod PAS 13395.95 inclusion body mtDod-seACP** PAS 19966.22 inclusion body Linker systems GSG: GGGGSGGGG (SEQ ID NO: 1) PAS: SPAAPAPASPAS (SEQ ID NO: 4) PASG: SPAAPAPASPASGGSG (SEQ ID GPAS: GGSGSPAAPAPASPAS (SEQ ID NO: 2) NO: 5) PAS2G: SPAAPAPASPASPAPSAPAASPA GPAS2: GGSGSPAAPAPASPASPAPSAP AGGSG (SEQ ID NO: 3) AASPAA (SEQ ID NO: 6) *MtDod-SpyC seems to be soluble in cellular environment, but formed yellow aggregates after cell lysis. **MtDod-seACP was not purified after refolding, because severe aggregation was observed during this step.

[0091] All constructs presented in Table 1, except mtDod-seACP, were obtained in good purity, as shown in FIG. 2.

[0092] The solubility and aggregation problems observed for some constructs may possibly be solved with formation of mtDod-heterododecamers, which allows reducing the density of attached entities on the surface. It has been demonstrated that such heterododecamers can be obtained with mtDod in vitro and in vivo. In a proof of concept approach, mtDod (WT), mtDod-His and mtDod-Strep were successfully assembled to heterododecamers. For in vitro heterododecamer formation, the mtDod (WT) and mtDod-Strep were jointly refolded, while for the formation of heterododecamers in vivo, several combinations of the three mtDod constructs were coexpressed. The high stability of the dodecamer allowed observing heterododecamers by SDS PAGE, which, owing to the differently sized mtDod constructs, made all subspecies visible that reflect the different compositions.

Example 2

[0093] A key feature of carrier proteins is a high stability that allows easy and prolonged storage. The high stability further enables the use of a wide range of conditions to conjugate cargos to the carrier. A cyclic thermal shift assay has been recently established, a termed thermocyclic fluorescence assay, to determine the stability of dodecins. This assay was applied to analyze the stability of mtDod constructs, and is based on the fluorescence quenching that is observed when flavins are bound to dodecin. In each binding pocket of the dodecamer, the two isoalloxazine ring systems of two bound flavins are embedded between symmetry-related tryptophans.[3,4,7]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 experiments, 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 5° C.). At the heating phase, FMN is released from the binding pocket and the fluorescence intensity increases. After the heating phase, the sample is cooled down, and FMN can rebind to the dodecamer (cooling phase) restoring initial low fluorescence values. As soon as the dodecamer denatures during heating and refolding is prevented in the cooling phase, 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 observed.

[0094] The thermocyclic fluorescence assay does only monitor the dodecameric stability, which, however, may be influenced by the attached cargo. As in PBS, all constructs, except mtDod-SZ1 and mtDod-SpyC, proved to be stable throughout the entire temperature range. The slightly destabilizing conditions of pH 4.2 were identified as suited to work out the impact of the cargo on the integrity of the mtDod dodecameric scaffold (see FIG. 3). Under this condition, mtDod-peptides denatured at 75-80° C. similar to mtDod wild type. The determination of the thermal stability of mtDod-proteins is more difficult, because the assay selectively addresses the mtDod dodecameric stability, but not the stability of the fused folds. Denaturation of the fused cargo may affect the FMN binding, although the dodecamer is correctly assembled, explaining the different curve shapes observed for some constructs (see FIG. 3). In screening temperatures for the heat denaturation of mtDod-mACP, for example the formation of yellow agglomerates above 55-60° C. is observed, indicating intact dodecamer that is capable of FMN binding. The high dodecameric stability of mtDod is also observed in SDS PAGE using the standard loading dye (2.5% SDS, pH 6.8). After staining, the monomer band became visible in the gel, but a dodecameric fraction remained as indicated by the high molecular weight band. In accordance to the lower stability at pH 4.2, observed in the thermocyclic fluorescence assay, a two component acidic loading dye was applied in SDS PAGE (3.3% SDS, pH 4.2 at heat treatment step, afterwards 2.5% SDS; pH 6.8) capable of fully denaturing the dodecamer. FIG. 2 shows the results of the SDS PAGE.

[0095] Depending on the use of the carrier matrix, storage conditions may be relevant, too. It was observed that most mtDod constructs can be frozen and thawed several times without significant aggregation. A glycerol containing buffer was used for the SpyCatcher constructs, and mtDod-PAS-msfGFP showed minor formation of green fluorescent aggregates.

Example 3

[0096] The accessibility and functionality of folds and peptides fused to mtDod were tested by the reactivity of the SpyTag/-Catcher pair (also SnoopTag/-Catcher pair). By equipping proteins/peptides with a short peptide (Tag) and a small protein fold (Catcher), that form a covalent bond upon interaction, proteins and/or peptides can be stably fused [8]. Applications range from attaching proteins from pathogens to scaffolds like virus like particles and IMX313 (heptamer forming coiled coils) for immunizations [9,10] to attaching enzymes to a scaffold in order to create enzyme hubs of increased catalytic efficiency [11].

[0097] For the SpyTag/-Catcher and SnoopTag/-Catcher reaction with the tagged mtDod constructs, seACP-SpyCatcher and mClover3-SnoopCatcher were prepared as cargo. For the inverse reaction of mtDod-SpyCatcher constructs, SpyTag-seACP was used as a cargo. For all reactions, two molar equivalents of cargo were used to promote the saturation of scaffold with cargo. The reactions were incubated for 20 h at 22° C. and analyzed by SDS PAGE. The results are shown in FIG. 4.

[0098] For all combinations of mtDod scaffold and cargo, the expected product band (or bands) of mtDod and the specific cargo (or two cargos) were observed in SDS PAGE. While for mtDod Spy-/SnoopTag constructs, no significant amount of unreacted scaffold proteins was observed, for the counterpart mtDod SpyCatcher constructs, bands of unreacted scaffold monomer were visible. It was shown that mtDod Spy-/SnoopTag constructs are lower in molecular mass as compared to the mtDod SpyCatcher constructs, and traces of unreacted scaffold protein may be less visible in SDS PAGE.

[0099] The data shows that a high degree of saturation was achieved, indicating that SpyTag/-Catcher or SnoopTag/-Catcher are accessible at the mtDod dodecamer scaffold and allowing the loading of cargo with high efficiency. For the double-tagged constructs SpyT-mtDod-SnpT or SnpT-mtDod-SpyT, heterovalently loaded with seACP-SpyCatcher and mClover3-SnoopCatcher, SDS PAGE reveals bands of single charged mtDod monomers. Since the surface density is increased by the double-tagging of mtDod, in this case the crowding of the scaffold surface seems to sterically limit the degree of conjugation with both cargos.

Example 4

[0100] A standard application of protein carriers lies in the production of antibodies against peptides or proteins [12]. Following the general procedure, the peptide or the protein of interest is linked to the carrier, usually BSA, keyhole limpet hemocyanin (KLH) or ovalbumin (OVA), by chemical ligation [12,13]. While the method is in general successful and widely used for antibody production, there can be problems related to the conjugation of antigen and carrier, like low stability of the conjugate or altered antigenic properties of the peptide.[14] 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. As demonstrated with an initial set of constructs, as schematically shown in FIG. 1, the mtDod scaffold can readily be expressed as soluble protein and easily purified by heat denaturation. In some cases, in vitro refolding of the dodecamer was required, because the constructs formed inclusion bodies. In order to evaluate the suitability of mtDod for antibody production, 11 fusion constructs, comprised of an N-terminal peptide, a linker PAS linker sequence and mtDod, were produced in E. coli and used for antibody production. The following table 2 shows the mtDod constructs used for the antibody production.

TABLE-US-00002 TABLE 2 MtDod constructs for antibody production. After purification by the heat treatment protocol constructs were verified by LCMS. calculated mass measured mass without start- by LCMS mtDod constructs Peptide sequence Met/Da (+1 H.sup.+)/Da mtDod-PAS-Pep 1 PKGGSGSGPTIEEVD (SEQ 10155 10156.7 ID NO: 7) mtDod-PAS-Pep2 PLEGDDDTSRMEEVD (SEQ 10434 10435.0 ID NO: 8) mtDod-PAS-Pep3 ECYPNEKNSVNMDLD (SEQ 10497 10803.4* ID NO: 9) mtDod-PAS-Pep4 VPSDSDKKLPEMDID (SEQ 10415 10416.1 ID NO: 10) mtDod-PAS-Pep5 DSSQHTKSSGEMEVD (SEQ 10363 10363.9 ID NO: 11) mtDod-PAS-Pep6 EQSTGQKRPLKNDEL (SEQ 10469 10470.2 ID NO: 12) mtDod-PAS-Pep7** ALMVYRCAPPRSSQF (SEQ 10453 — ID NO: 13) mtDod-PAS-Pep8 LVTGESLEQLRRGLA (SEQ 10368 10369.1 ID NO: 14) mtDod-PAS-Pep9 MKGKEEKEGGARLGA (SEQ 10287 10288.0 ID NO: 15) mtDod-PAS-Pep10 EERRIHQESE (SEQ ID NO: 10038 10039.6 16) mtDod-PAS-Pep11 NHEGDEDDSH (SEQ ID NO:  9880  9881.3 17) mtDod-PAS-H7 HHHHHHH (SEQ ID NO: 18)  9704  9705.2 *Difference of mass is about 305 Da and could be caused by S-glutathionylation (15, 16). No mass for the unmodified mtDod-PAS-Pep3 was observed. **mtDod-PAS-Pep7 formed inclusion bodies and was not further purified.

[0101] For cloning, the Peptide-encoding sequence was provided on primer sequences and introduced in a single step by ligation free cloning methods. Recombinant expressions and purifications followed the established protocols. All constructs were received as soluble proteins, except mtDod-PAS-Pep7 that formed inclusion bodies. The yellow color of the inclusion bodies indicated assembled dodecamer and aggregation most likely induced by disulfide-bridges formation by the cysteine in the Pep7 sequence. All constructs, except mtDod-PAS-Pep7, were further purified by two cycles of DMSO-induced precipitations. FMN was added before all constructs were finally purified by SEC to remove unbound FMN and remaining DMSO as well as to select for dodecameric species. For example, the SEC profiles of FMN:mtDod-PAS-Pep1 is shown in FIG. 6a. FMN-saturated mtDod constructs (FMN:mtDod constructs) can be determined in concentration by absorbance at 450 nm and are susceptible to stability measurements by the thermocyclic fluorescence assay. All constructs were received as dodecamers as indicated by SEC. MtDod-PAS-Pep3 shows in addition to the dodecamer species higher oligomeric states in SEC. The dodecamer containing fractions were pooled and the purity was controlled by SDS-PAGE. FIG. 6b shows the thermocyclic fluorescence assay of mtDod-PAS-Pep constructs. Molecular masses of the constructs were verified with LCMS. The above mentioned Table 2 shows the results.

[0102] The yield of each mtDod construct was between 150-500 mg per liter E. coli expression culture (values extrapolated as only a fraction was purified, absolute yields 20-50 mg). With 20 mg, MtDod-PAS-Pep3 was received in the lowest yield, which may originate from aggregation of dodecamers, induced by disulfide bridges formed by a cysteine in the Pep3 sequence. In general, the constructs mtDod-PAS-Pep3 and mtDod-PAS-Pep7 indicate that cysteine containing peptides can cause problems when processed via the described purification strategy, owing to the oxidative conditions imposed by FMN. Accordingly, a changed protocol that avoids working with FMN in excess and/or that includes reducing agents should make those constructs accessible. The thermocyclic fluorescence assay, showed the high thermal stability of all mtDod-PAS-Pep constructs, similar as the wild type [4]. The results are shown in FIG. 6c.

[0103] Endotoxin concentrations, measured in endotoxin units (EU) via a Limulus amebocyte lysate (LAL) test, were determined to avoid an endotoxin shock in immunizations. Dod-PAS-Pep3 and Dod-PAS-Pep6 contained the highest amount of endotoxin with 73 EU/mg and 55 EU/mg, respectively; all other samples showed values less than 30 U/mg (average of all constructs 30±23 U/mg). Since less than 0.1 mg protein was used per injection, none of the samples were in the critical range to cause an endotoxin shock (The non-pyrogenic amount of endotoxin is less than 5 EU/kg. A rabbit used for immunization weighs on average 5 kg, so the amount per injection should be less than 25 EU (=0.25 ng LPS/ml)) [15, 16]. Antibodies were produced in rabbits, and immunizations conducted with 5 boosts during 63 days and using adjuvants MF59/AddaVax or Montanide ISA 51. The antibodies were purified by affinity chromatography with the respective Dod-PAS-Pep construct immobilized on the column matrix. For the 10 Dod-PAS-Pep constructs subjected to immunizations, purified antibodies were obtained. 6 of the 10 antibodies recognized the peptide containing target protein either as heterologously expressed protein or as protein part of HEK293T lysate. FIG. 7a shows that the antibodies overall showed no systematic off/background targeting in the used lysates, indicating that mtDod-PAS is a suitable matrix in this respect. The 4 remaining antibodies (constructs) did not recognize the peptide containing target protein and only recognize the mtDod-PAS matrix and the respective mtDod-PAS construct. In Western Blotting, the target recognizing antibodies performed comparably to available commercial antibodies. This is shown in FIG. 7b. For all target-recognizing antibodies, the recognition limit/range of purified recombinant protein was analyzed and compared with commercially available antibodies.

TABLE-US-00003 TABLE 3 Recognition strength of mtDod-PAS-Pep construct antibodies. The lowest amount of loaded purified recombinant protein (in ng) that could be clearly labeled with the respective antibody was used as detection limit. MtDod-PAS-Pep construct derived antibodies were used at a final concentration of 1 μg/mL and commercial antibodies at the recommended dilution. For comparison the exposure time for each antibody pair (mtDod-PAS-Pep construct derived and commercial) was the same, while it was varied for the different pairs. lowest amount of recombinant antibody derived protein clearly labelled/ng form mtDod target site of derived commercial construct the antibody antibody antibody mtDod-PAS-Pep 1 HSP70 125-250  60-125 c-terminal mtDod-PAS-Pep2 HSP90 125-250 — c-terminal mtDod-PAS-Pep3 HSP110 60 250-500 c-terminal mtDod-PAS-Pep4 H2  60-125 125-250 c-terminal mtDod-PAS-Pep5 H3 500* — c-terminal mtDod-PAS-Pep6 H4 — — c-terminal mtDod-PAS-Pep8 DTR — — mtDod-PAS-Pep9 CHIP 125-250 60 N-term mtDod-PAS-Pep10 CHIP  —** — broken helix mtDod-PAS-Pep11 CHIP  60-125 60 tip of helix *mtDod-PAS-Pep5 derived antibodies showed only a very weak signal for 1 μg and 500 μg of recombinant protein with no intensity difference. While something is recognized the antibody preparation was counted as not target recognizing. **mtDod-PAS-Pep10 derived antibodies didn't recognize purified CHIP, but seem to recognize a protein in CHIP overexpressing cells, no detection range was determined.

[0104] The mtDod-PAS-Pep derived antibodies show that mtDod-PAS is suitable as a carrier system for the production of peptide specific antibodies. Key benefits of mtDod-PAS as a carrier are the easy cloning, uncomplicated production/purification and the high yields. Problems in the purification of constructs that occur from the oxidative conditions caused by the high FMN concentration; i.e., promoting disulfide formation between exposed cysteines of the peptide tags, may be avoided when working under reducing conditions.

Example 5

[0105] This example relates to tests using a dodecin-carrier that has been modified with two epitopes A and B derived from Slc36a3. Slc36a3 is a trans-membrane protein exclusively expressed in elongated spermatids that are easily recognizable by the drop-like shape of their nuclei. The results (see FIG. 8) show that the antibodies label the expected cells and thus are specific. Antibodies derived from KLH epitope conjugates (FIG. 8, left side; 324 and 325) produce overall a higher background signal, likely through unspecific binding, in contrast to antibodies derived from mtDod epitope fusion constructs (right side; SAO359 and SAO360).

[0106] All antibodies were purified by a standard protein A purification strategy from serum of immunized rabbits. Rabbits were immunized via 4 injections of the respective antigen (each 200 μg) over 56 days and serum was collected after 87 days. Immunization and serum collection were conducted by Eurogentec, Belgium. “C+” indicates that a cysteine was added at the N-terminus of the peptide for crosslinking. For dodecin constructs the added cysteines were removed. Crosslinking agent was MBS.

Peptides as used for KLH epitope conjugates (same set of epitopes)

TABLE-US-00004 324 (KLH mixture): (SEQ ID NO: 20) C+MKFGTDTQASIT and (SEQ ID NO: 21) C+QQTHFYMANSTRVHI 325 (KLH mixture): (SEQ ID NO: 20) C+MKFGTDTQASIT and (SEQ ID NO: 21) C+QQTHFYMANSTRVHI
Peptides as used for MtDod epitope fusion constructs:

TABLE-US-00005 SAO359 (mtDod mixture): (SEQ ID NO: 20) mtDod-PAS-MKFGTDTQASIT and (SEQ ID NO: 21) mtDod-PAS-QQTHFYMANSTRVHI SAO360 (mtDod N- and C-terminal): (SEQ ID NO: 20) S+MKFGTDTQASIT  (SEQ ID NO: 21) -nPAS-mtDod-PAS-QQTHFYMANSTRVHI
“S+”: serine added to the epitope to avoid cleavage of the N-terminal methionine of the epitope.
Linker: nPAS: SAPSAPAPAAPSPAS (SEQ ID NO: 19); PAS: SPAAPAPASPAS (SEQ ID NO: 4)

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