Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA_31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm², whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm² was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.     

Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.     

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

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.     

An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology

The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.     

Exploitation of the S-layer self-assembly system for site directed immobilization of enzymes demonstrated for an extremophilic laminarinase from Pyrococcus furiosus

A fusion protein based on the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and the enzyme laminarinase (LamA) from Pyrococcus furiosus was designed and overexpressed in Escherichia coli. Due to the construction principle, the S-layer fusion protein fully retained the self-assembly capability of the S-layer moiety, while the catalytic domain of LamA remained exposed at the outer surface of the formed protein lattice. The enzyme activity of the S-layer fusion protein monolayer obtained upon recrystallization on silicon wafers, glass slides and different types of polymer membranes was determined colorimetrically and related to the activity of sole LamA that has been immobilized with conventional techniques. LamA aligned within the S-layer fusion protein lattice in a periodic and orientated fashion catalyzed twice the glucose release from the laminarin polysaccharide substrate in comparison to the randomly immobilized enzyme. In combination with the good shelf-life and the high resistance towards temperature and diverse chemicals, these novel composites are regarded a promising approach for site-directed enzyme immobilization.     

Display of a peptide mimotope on a crystalline bacterial cell surface layer (S-layer) lattice for diagnosis of Epstein-Barr virus infection

Fusion proteins based on the crystalline bacterial cell surface layer (S-layer) proteins SbpA from Bacillus sphaericus CCM 2177 and SbsB from Geobacillus stearothermophilus PV72/p2 and a peptide mimotope F1 that mimics an immunodominant epitope of Epstein-Barr virus (EBV) were designed and overexpressed in Escherichia coli. Constructs were designed such that the peptide mimotope was presented either at the C-terminus (SbpA/F1) or at the N-terminus (SbsB/F1) of the respective S-layer proteins. The resulting S-layer fusion proteins, SbpA/F1 and SbsB/F1, fully retained the intrinsic self-assembly capability of the S-layer moiety into monomolecular lattices. As determined by immunodot assays and ELISAs using monoclonal antibodies, the F1 mimotope was well-presented on the outer surface of the S-layer lattices and accessible for antibody binding. Further comparison of the two S-layer fusion proteins showed that the S-layer fusion protein SbpA/F1 had a higher antibody binding capacity than SbsB/F1 in aqueous solution and in immune sera, illustrating the importance of epitope orientation on the performance of solid-phase immunoassays. To assess the diagnostic values of S-layer mimotope fusion protein SbpA/F1, we screened a panel of 83 individual EBV IgM-positive, EBV negative, and potential cross-reactive sera for their reactivities. This resulted in 98.2% specificity and 89.3% sensitivity, and furthermore no cross-reactivity with related viral disease states including rheumatoid factor was observed. This study shows the potential of S-layer fusion proteins as a matrix for site-directed immobilization of small ligands in solid-phase immunoassays using EBV diagnostics as a model system.     

Molecular characterization of the S-layer gene,sbpA, of Bacillus sphaericus CCM 2177 and production of a functional S-layer fusion protein with the ability to recrystallize in a defined orientation while presenting the fused allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

Molecular Characterization of the S-Layer Gene, sbpA, of Bacillus sphaericus CCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5′ end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA_31-1068). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA_31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA_31-1068/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.     

Identification by flagellum display of an epithelial cell- and fibronectin-binding function in the SlpA surface protein of Lactobacillus brevis

Depletion of the SlpA protein from the bacterial surface greatly reduced the adhesion of Lactobacillus brevis ATCC 8287 to the human intestinal cell lines Caco-2 and Intestine 407, the endothelial cell line EA-hy926, and the urinary bladder cell line T24, as well as immobilized fibronectin. For functional analysis of the SlpA surface protein, different regions of the slpA gene were expressed as internal in-frame fusions in the variable region of the fliC(H7) gene of Escherichia coli. The resulting chimeric flagella carried inserts up to 275 amino acids long from the mature S-layer protein, which is 435 amino acids in size. The expression of the SlpA fragments on the chimeric flagella was assessed by immunoelectron microscopy and Western blotting using anti-SlpA antibodies, and their binding to human cells was assessed by indirect immunofluorescence. Chimeric flagella harboring inserts that represented the N-terminal part of the S-layer protein bound to the epithelial cell lines, whereas the C-terminal part of the S-layer protein did not confer binding on the flagella. The shortest S-layer peptide capable of detectable binding was 81 amino acid residues in size and represented residues 96 through 176 in the unprocessed S-layer protein. The bacteria and the chimeric flagella did not show detectable binding to erythrocytes, whereas the SlpA-expressing ATCC 8287 cells as well as the chimeric SlpA 96-245/FliC flagella bound to immobilized fibronectin. The N-terminal SlpA peptide 96-176 or 96-200 fused to FliC was not recognized in Western blotting or immunoelectron microscopy by a polyclonal serum raised against the S-layer protein; the antiserum, however, reacted in immunofluorescence with the ATCC 8287 cells. In contrast, an antiserum raised against the His-tagged peptide 96-245 of SlpA bound to the hybrid flagella with the N-terminal SlpA inserts but did not react with ATCC 8287 cells. The results identify the S-layer of L. brevis ATCC 8287 as an adhesin with affinity for human epithelial cells and fibronectin and locate the receptor-binding region within a fragment of 81 amino acids in the N-terminal part of the molecule, which in native S-layer seems inaccessible to antibodies.     

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.     

Relevance of charged groups for the integrity of the S-layer from Bacillus coagulans E38-66 and for molecular interactions.

In this paper, the importance of charged amino and carboxyl groups for the integrity of the cell surface layer (S-layer) lattice from Bacillus coagulans E38-66 and for the self-assembly of the isolated subunits was investigated. Amidination of the free amino groups which preserved their positive net charge had no influence on both. On the other hand, acetylation and succinylation, which converted the amino groups into either neutral or negatively charged groups, and amidation of carboxyl groups were accompanied by the disintegration or at least by the loss of the regular structure of the S-layer lattice. Treatment of S-layer monolayers with the zero-length cross-linker carbodiimide led to the introduction of peptide bonds between activated carboxyl groups and amino groups from adjacent subunits. This clearly indicated that in the native S-layer lattice the charged groups are located closely enough for direct electrostatic interactions. Under disrupting conditions in which the S-layer polypeptide chains were unfolded, 58% of the Asx and Glx residues could be amidated, indicating that they occur in the free carboxylic acid form. As derived from chemical modification of monolayer self-assembly products, about 90% of the lysine and 70% of the aspartic and glutamic acid residues are aligned on the surface of the S-layer protein domains. This corresponded to 45 amino groups and to 63 carboxyl groups per S-layer subunit. Labelling experiments with macromolecules with different sizes and charges and adsorption studies with ion-exchange particles revealed a surplus of free carboxyl groups on the inner and on the outer faces of the S-layer lattice. Since the carboxyl groups on the outer S-layer face were accessible only for protein molecules significantly smaller then the S-layer protomers or for positively charged, thin polymer chains extending from the surface of ion-exchange beads, the negatively charged sites must be located within indentations of the corrugated S-layer protein network. This was in contrast to the carboxyl groups on the inner S-layer face, which were found to be exposed on elevations of the S-layer protein domains (D. Pum, M. Sára, and U.B. Sleytr, J. Bacteriol. 171:5296-5303, 1989).     

Evidence that the N-terminal part of the S-layer protein from Bacillus stearothermophilus PV72/p2 recognizes a secondary cell wall polymer.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1gamma chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.     

The S-Layer Proteins of Two Bacillus stearothermophilus Wild-Type Strains Are Bound via Their N-Terminal Region to a Secondary Cell Wall Polymer of Identical Chemical Composition

Two Bacillus stearothermophilus wild-type strains were investigated regarding a common recognition and binding mechanism between the S-layer protein and the underlying cell envelope layer. The S-layer protein from B. stearothermophilus PV72/p6 has a molecular weight of 130,000 and assembles into a hexagonally ordered lattice. The S-layer from B. stearothermophilus ATCC 12980 shows oblique lattice symmetry and is composed of subunits with a molecular weight of 122,000. Immunoblotting, peptide mapping, N-terminal sequencing of the whole S-layer protein from B. stearothermophilus ATCC 12980 and of proteolytic cleavage fragments, and comparison with the S-layer protein from B. stearothermophilus PV72/p6 revealed that the two S-layer proteins have identical N-terminal regions but no other extended structurally homologous domains. In contrast to the heterogeneity observed for the S-layer proteins, the secondary cell wall polymer isolated from peptidoglycan-containing sacculi of the different strains showed identical chemical compositions and comparable molecular weights. The S-layer proteins could bind and recrystallize into the appropriate lattice type on native peptidoglycan-containing sacculi from both organisms but not on those extracted with hydrofluoric acid, leading to peptidoglycan of the A1γ chemotype. Affinity studies showed that only proteolytic cleavage fragments possessing the complete N terminus of the mature S-layer proteins recognized native peptidoglycan-containing sacculi as binding sites or could associate with the isolated secondary cell wall polymer, while proteolytic cleavage fragments missing the N-terminal region remained unbound. From the results obtained in this study, it can be concluded that S-layer proteins from B. stearothermophilus wild-type strains possess an identical N-terminal region which is responsible for anchoring the S-layer subunits to a secondary cell wall polymer of identical chemical composition.     

Affinity cross-flow filtration: purification of IgG with a novel protein a affinity matrix prepared from two-dimensional protein crystals

In this article, we describe the use of 1- to 2-mum sized affinity microparticles for the isolation and purification of IgG from artificial IgG-human serum albumin mixtures and clarified hybridoma cell culture supernatants by affinity cross-flow filtration. Affinity microparticles were prepared from cell wall fragments of Clostridium thermohydrosulfuricum L111-69, in which the peptidoglycan-containing layer was completely covered with a hexagonally ordered S-layer lattice. After crosslinking the S-layer protein with glutaraldehyde, carboxyl groups from acidic amino acids were activated with carbodiimide and used for immobilization of Protein. A. Quantitative determination confirmed that Protein A molecules formed a monomolecular layer on the outermost surface of the S-layer lattice. Affinity microparticles were found to withstand high centrifugal and shear forces and revealed no Protein A leakage or S-layer protein release under cross-flow conditions between pH 2 to 12. The IgG-binding capacity of affinity microparticles was investigated under crossflow conditions and compared with that obtained in batch adsorption processes. (c) 1994 John Wiley & Sons, Inc.     

Immobilization of Saccharomyces cerevisiae cells to protein G-Sepharose by cell wall engineering

In this work, we explored the possibility of using the targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae, by fusing it to a cell wall protein, to construct yeast strains whose cells display on their surface proteins that bind to a matrix, so as to achieve the immobilization of the whole cells. With this aim, we created a gene fusion that comprises the region responsible for attachment of a cell wall protein to the cell wall, and the IgG binding region of staphylococcal protein A, and expressed it in the mnn1mnn9 strain of S. cerevisiae. The surface display of the protein A-Icwp fusion protein was positively monitored; however, direct immobilization of the cells on an IgG-Sepharose matrix did not produce the expected results, probably due to the fusion protein not being completely exposed on the surface of the cells. To solve this problem we incubated the cells first with rabbit preimmune serum and then with goat anti-rabbit IgGs, so as to create a complex (yeast cell-protein A-rabbit IgG-goat IgG). Cells treated in this way were successfully immobilized on a protein G-Sepharose matrix, due to the binding properties of goat IgGs to streptococcal protein G.     

Recombinant Escherichia coli strain produces a ZZ domain displaying biopolyester granules suitable for immunoglobulin G purification

The immunoglobulin G (IgG) binding ZZ domain of protein A from Staphylococcus aureus was fused to the N terminus of the polyhydroxyalkanoate (PHA) synthase from Cupriavidus necator. The fusion protein was confirmed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry and mediated formation of ZZ domain-displaying PHA granules in recombinant Escherichia coli. The IgG binding capacity of isolated granules was assessed using enzyme-linked immunosorbent assay and could be enhanced by the overproduction of the ZZ-PHA synthase. ZZ-PHA granules enabled efficient purification of IgG from human serum.     

Expression of an endotoxin-free S-layer/allergen fusion protein in gram-positive <Emphasis Type="Italic">Bacillus subtilis</Emphasis> 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.     

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium

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.     

Ellipsometric study of immunologic reactions on the surface of silicon

Adsorption of human serum albumin (HSA), egg albumin (EA), immunoglobulin G (IgG) and immunologic reactions occurring between them on silicon surface were studied by ellipsometry. Adsorption of HSA, IgG and antibodies on the monolayer of antigen is monomolecular in their isoelectric points and can be depicted by Langmuir's equation. Adsorption of EA is polymolecular apparently because of great tendency of the protein to aggregation in aqueous solutions. Comparison of the magnitudes of the protein monolayer thickness and areas per adsorbed molecule with their linear dimensions indicate that they preserve their native conformation. This allows an evaluation of the maximum number of the active sites (as approximately four) on the antigen surface accessible for antibodies and the adsorption constants for specific and nonspecific adsorption of IgG.     

Structure, surface charge, and self-assembly of the S-layer lattice from Bacillus coagulans E38-66.

In freeze-etched preparations, whole cells from Bacillus coagulans E38-66 exhibited an oblique S-layer lattice (a = 9.4 nm; b = 7.4 nm; gamma = 80 degrees). The three-dimensional structure of the crystalline array was characterized by optical and computer image analysis. The lattice showed two distinctly shaped types of pores. In vitro self-assembly of isolated subunits yielded flat sheets and open-ended cylinders composed of two back-to-back monolayers. Unlike whole cells, in vitro self-assembly products were capable of binding polycationized ferritin (pI, approximately 11). This showed that only the inner S-layer face adhering to the peptidoglycan-containing layer in whole cells was net negatively charged. S-layer monomers and/or oligomers were capable of generating a closed monolayer with oblique symmetry on poly-L-lysine-coated supports. The monolayer had a typical crazy paving appearance, with numerous crystal boundaries. The handedness of the oblique lattice and ability to bind polycationized ferritin revealed that the subunits had bound with the outer, not net negatively charged face to the poly-L-lysine-coated supports. Carbodiimide-activated carboxyl groups on either cell wall fragments or self-assembly products could covalently bind high-molecular-weight nucleophiles such as ferritin. This confirmed the location of negatively charged carboxyl groups on the outermost surface of both S-layer faces. The difference in pH optimum for carbodiimide activation indicated a preponderance of alpha- and beta-carboxyl groups on the inner S-layer face and a preponderance of beta- and gamma-carboxyl groups on the outer S-layer face.