A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

Breitwieser, Andreas; Egelseer, Eva M.; Moll, Wulf-Dieter; Ilk, Nicola; Hotzy, Christoph; Bohle, Barbara; Ebner, Christof; Sleytr, Uwe B.; Sára, Margit

doi:10.1093/protein/15.3.243

Cited by 53 publications

(41 citation statements)

References 31 publications

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“…7) indicates that the C-terminal HA tag is exposed to the ambient environment, and Fourier transformation analysis revealed the formation of lattices with lattice spacing corresponding to that of native SslA of 13.2 nm (32). The results obtained by immunolabeling of selfassembly products are in good agreement with studies on various S-layer fusion proteins that exhibit C-terminally fused functional domains (e.g., streptavidin [2,25], birch pollen allergen [8,15,18], camel antibody [29], single amino acids [2], and enhanced green fluorescent protein [eGFP] [16]). Studies on structurefunction relationships revealed that the middle and, in some cases, the C-terminal parts of S-layer proteins are responsible for selfassembly into 2-dimensional protein arrays.…”

Section: Discussionsupporting

confidence: 82%

Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Knobloch

Rödel

2012

Appl Environ Microbiol

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Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N-and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.

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Section: Discussionsupporting

confidence: 82%

Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Knobloch

Rödel

2012

Appl Environ Microbiol

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“…A chromosomal integration, utilizing the S-layer homologous motifs of the Bacillus anthracis S-layer proteins EA1 or Sap fused with levansucrase of B. subtilis, resulted in anchoring of the fusion protein to the bacterial cell surface (30). There are also reports on several non-Lactobacillus S-layer fusion proteins for which the protein production is plasmid derived either in a heterologous host (5,49) or in a null mutant lacking the wild-type S-layer protein gene (2). Our work is thus the first to describe the construction of a chromosomally encoded S-layer fusion protein, utilizing the entire S-layer protein gene.…”

Section: Discussionmentioning

confidence: 99%

Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer

Åvall-Jääskeläinen

Kylä-Nikkilä

Kahala

et al. 2002

Appl Environ Microbiol

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So far, the inability to establish viable Lactobacillus surface layer (S-layer) null mutants has hampered the biotechnological applications of Lactobacillus S-layers. In this study, we demonstrate the utilization of Lactobacillus brevis S-layer subunits (SlpA) for the surface display of foreign antigenic epitopes. With an inducible expression system, L. brevis strains producing chimeric S-layers were obtained after testing of four insertion sites in the slpA gene for poliovirus epitope VP1, that comprises 10 amino acids. The epitope insertion site allowing the best surface expression was used for the construction of an integration vector carrying the gene region encoding the c-Myc epitopes from the human c-myc proto-oncogene, which is composed of 11 amino acids. A gene replacement system was optimized for L. brevis and used for the replacement of the wild-type slpA gene with the slpA-c-myc construct. A uniform S-layer, displaying on its surface the desired antigen in all of the S-layer protein subunits, was obtained. The success of the gene replacement and expression of the uniform SlpA-c-Myc recombinant S-layer was confirmed by PCR, Southern blotting MALDI-TOF mass spectrometry, whole-cell enzyme-linked immunosorbent assay, and immunofluorescence microscopy. Furthermore, the integrity of the recombinant S-layer was studied by electron microscopy, which indicated that the S-layer lattice structure was not affected by the presence of c-Myc epitopes. To our knowledge, this is the first successful expression of foreign epitopes in every S-layer subunit of a Lactobacillus S-layer while still maintaining the S-layer lattice structure.Many organisms from the domains Bacteria and Archaea possess a surface layer (S-layer) as the outermost structure of the cell envelope. S-layers are composed of regularly arranged proteinaceous subunits of a single protein or glycoprotein species with molecular masses ranging from 40 to 170 kDa (43, 48). S-layer proteins represent 10 to 15% of the total protein of the bacterial cell, and S-layer lattices cover the cell surface during all stages of growth, which indicates that efficient gene expression, S-layer protein synthesis, and secretion take place (4). A high content of hydrophobic and acidic amino acids and a low theoretical isoelectric point (pI) are typical features of S-layer proteins (48). In contrast, very high pI values have been described for the S-layer proteins from various lactobacilli (4, 54) and Methanothermus fervidus (6). In general, S-layers have been considered to function as cell shape determinants, protective coats, promoters for cell adhesion and surface recognition, and molecular and ion traps; however, no general function found in all S-layers has been recognized (47, 48).S-layers have been found from many species of the genus Lactobacillus (31, 56). However, the S-layer protein genes have been cloned and sequenced only from Lactobacillus brevis ATCC 8287 (54) and from the closely related species L. acidophilus (3), L. helveticus (8), and L. crispatus (46). Th...

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“…The study was approved by the local medical ethics committee (Vienna, Austria). RSbsC-Bet v 1 and rSbsC were produced, as described (30). Endotoxin levels in rSbsC-Bet v 1 and rSbsC were below 0.4 EU/mg, as determined by Limulus amebocyte lysate assay (BioWhittaker).…”

Section: Patients Allergens and Reagentsmentioning

confidence: 99%

“…We have proposed to engineer allergy vaccines by genetic fusion of allergens with S-layer proteins: the major birch pollen allergen Bet v 1 was fused with the S-layer protein from the nonpathogenic bacteria Geobacillus stearothermophilus ATCC 12980 (30,31). The resulting allergen-S-layer fusion protein rSbsC-Bet v 1 induced IFN-␥ and IL-10 production in PBMC and Bet v 1-specific Th2 clones from birch pollen-allergic donors (31).…”

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confidence: 99%

A Novel Approach to Specific Allergy Treatment: The Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1 Matures Dendritic Cells That Prime Th0/Th1 and IL-10-Producing Regulatory T Cells

Gerstmayr

Ilk

Schabussova

et al. 2007

The Journal of Immunology

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An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity, induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and induced IFN-γ and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Bet v 1 on immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4+ T lymphocytes isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced the differentiation of naive T cells into IFN-γ-producing cells. This effect was IL-12 dependent. In parallel, a substantial number of naive T cells developed into IL-10-producing CD25+Foxp3+CLTA-4+ cells capable of active suppression. Thus, rSbsC-Bet v 1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach to improve vaccines for specific immunotherapy of atopic allergies.

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A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

Cited by 53 publications

References 31 publications

Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Production, Secretion, and Cell Surface Display of Recombinant Sporosarcina ureae S-Layer Fusion Proteins in Bacillus megaterium

Surface Display of Foreign Epitopes on the Lactobacillus brevis S-Layer

A Novel Approach to Specific Allergy Treatment: The Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1 Matures Dendritic Cells That Prime Th0/Th1 and IL-10-Producing Regulatory T Cells

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