S-layers: principles and applications.

Monomolecular arrays of protein or glycoprotein subunits forming surface layers (S-layers) are one of the most commonly observed prokaryotic cell envelope components. S-layers are generally the most abundantly expressed proteins, have been observed in species of nearly every taxonomical group of walled bacteria, and represent an almost universal feature of archaeal envelopes. The isoporous lattices completely covering the cell surface provide organisms with various selection advantages including functioning as protective coats, molecular sieves and ion traps, as structures involved in surface recognition and cell adhesion, and as antifouling layers. S-layers are also identified to contribute to virulence when present as a structural component of pathogens. In Archaea, most of which possess S-layers as exclusive wall component, they are involved in determining cell shape and cell division. Studies on structure, chemistry, genetics, assembly, function, and evolutionary relationship of S-layers revealed considerable application potential in (nano)biotechnology, biomimetics, biomedicine, and synthetic biology.

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.

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.

Identifying assembly-inhibiting and assembly-tolerant sites in the SbsB S-layer protein from Geobacillus stearothermophilus.

Surface layer (S-layer) proteins self-assemble into two-dimensional crystalline lattices that cover the cell wall of all archaea and many bacteria. We have generated assembly-negative protein variants of high solubility that will facilitate high-resolution structure determination. Assembly-negative versions of the S-layer protein SbsB from Geobacillus stearothermophilus PV72/p2 were obtained using an insertion mutagenesis screen. The haemagglutinin epitope tag was inserted at 23 amino acid positions known to be located on the monomer protein surface from a previous cysteine accessibility screen. Limited proteolysis, circular dichroism, and fluorescence were used to probe whether the epitope insertion affected the secondary and tertiary structures of the monomer, while electron microscopy and size-exclusion chromatography were employed to examine proteins' ability to self-assemble. The screen not only identified assembly-compromised mutants with native fold but also yielded correctly folded, self-assembling mutants suitable for displaying epitopes for biomedical and biophysical applications, as well as cryo-electron microscopy imaging. Our study marks an important step in the analysis of the S-layer structure. In addition, the approach of concerted insertion and cysteine mutagenesis can likely be applied for other supramolecular assemblies.

Genetic engineering of the S-layer protein SbpA of Lysinibacillus sphaericus CCM 2177 for the generation of functionalized nanoarrays.

The mesophilic organism Lysinibacillus sphaericus CCM 2177 produces the surface (S)-layer protein SbpA, which after secretion completely covers the cell surface with a crystalline array exhibiting square lattice symmetry. Because of its excellent in vitro recrystallization properties on solid supports, SbpA represents a suitable candidate for genetically engineering to create a versatile self-assembly system for the development of a molecular construction kit for nanobiotechnological applications. The first goal of this study was to investigate the surface location of 3 different C-terminal amino acid positions within the S-layer lattice formed by SbpA. Therefore, three derivatives of SbpA were constructed, in which 90, 173, or 200 C-terminal amino acids were deleted, and the sequence encoding the short affinity tag Strep-tag II as well as a single cysteine residue were fused to their C-terminal end. Recrystallization studies of the rSbpA/STII/Cys fusion proteins indicated that C-terminal truncation and functionalization of the S-layer protein did not interfere with the self-assembly capability. Fluorescent labeling demonstrated that the orientation of the crystalline rSbpA(31-1178)/STII/Cys lattice on solid supports was the same, like the orientation of wild-type S-layer protein SbpA on the bacterial cell. In soluble and recrystallized rSbpA/STII/Cys fusion proteins, Strep-tag II was used for prescreening of the surface accessibility, whereas the thiol group of the end-standing cysteine residue was exploited for site-directed chemical linkage of differently sized preactivated macromolecules via heterobifunctional cross-linkers. Finally, functionalized two-dimensional S-layer lattices formed by rSbpA(31-1178)/STII/Cys exhibiting highly accessible cysteine residues in a well-defined arrangement on the surface were utilized for the template-assisted patterning of gold nanoparticles.