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Background  Genetic fusion of the major birch pollen allergen (Bet v1) to bacterial surface-(S)-layer proteins resulted in recombinant proteins exhibiting reduced allergenicity as well as immunomodulatory capacity. Thus, S-layer/allergen fusion proteins were considered as suitable carriers for new immunotherapeutical vaccines for treatment of Type I hypersensitivity. Up to now, endotoxin contamination of the fusion protein which occurred after isolation from the gram-negative expression host  E. coli  had to be removed by an expensive and time consuming procedure. In the present study, in order to achieve expression of pyrogen-free, recombinant S-layer/allergen fusion protein and to study the secretion of a protein capable to self-assemble, the S-layer/allergen fusion protein rSbpA/Bet v1 was produced in the gram-positive organism  Bacillus subtilis  1012.    Results  The chimaeric gene encoding the S-layer protein SbpA of  Lysinibacillus sphaericus  CCM 2177 as well as Bet v1 was cloned and expressed in  B. subtilis  1012. For that purpose, the  E. coli-B. subtilis  shuttle vectors pHT01 for expression in the  B. subtilis  cytoplasm and pHT43 for secretion of the recombinant fusion protein into the culture medium were used. As shown by western blot analysis, immediately after induction of expression,  B. subtilis  1012 was able to secret rSbpA/Bet v1 mediated by the signal peptide amyQ of  Bacillus amyloliquefaciens . Electron microscopical investigation of the culture medium revealed that the secreted fusion protein was able to form self-assembly products in suspension but did not recrystallize on the surface of the  B. subtilis  cells. The specific binding mechanism between the N-terminus of the S-layer protein and a secondary cell wall polymer (SCWP), located in the peptidoglycan-containing sacculi of  Ly. sphaericus  CCM 2177, could be used for isolation and purification of the secreted fusion protein from the culture medium. Immune reactivity of rSbpA/Bet v1 could be demonstrated in immunoblotting experiments with Bet v1 specific IgE containing serum samples from patients suffering birch pollen allergy.    Conclusions  The impact of this study can be seen in the usage of a gram-positive organism for the production of pyrogen-free self-assembling recombinant S-layer/allergen fusion protein with great relevance for the development of vaccines for immunotherapy of atopic allergy.

microbial cell factories

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive Bacillus subtilis 1012 for the potential application as vaccines for immunotherapy of atopic allergy