Margit Sára

S-Layer proteins.

Cell walls are an important structural component of prokaryotic organisms and essential for many aspects of their life. Particularly, the diverse structures of the outermost boundary layers strongly reflect adaptations of organisms to specific ecological and environmental conditions ([6][1]). Over

S-layer-streptavidin fusion proteins as template for nanopatterned molecular arrays.

Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding d-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

Bacterial and archaeal S-layer proteins: structure-function relationships and their biotechnological applications.

Crystalline cell surface layers (S-layers) composed of planar assemblies of protein or glycoprotein subunits are one of the most commonly observed cell envelope structures of bacteria and archaea. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at liquid-surface interfaces, including lipid films, on liposomes and on solid supports. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattices are aligned in well-defined positions and orientations. These characteristic features of S-layers have led to various applications in biotechnology, vaccine development, diagnostics, biomimetics and molecular nanotechnology.

Crystalline bacterial cell surface layers

Crystalline arrays of proteinaceous subunits forming surface layers (S‐layers) are one of the most commonly observed prokaryotic cell envelope structures. They are ubiquitous amongst Gram‐positive and Gram‐negative archaeobacteria and eubacteria and, if present, account for the major protein species produced by the cells. S‐layers can provide organisms with a selection advantage by providing various functions including protective coats, molecular sieves, ion traps and structures involved in cell surface interactions. S‐layers were identified as contributing to virulence when present as a structural component of pathogens. In Gram‐negative archaeobacteria they are involved in determining cell shape and cell division. The crystalline arrays reveal a broad‐application potential in biotechnology, vaccine development and molecular nanotechnology.

V. Functions of S‐layers

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

Characterization and use of crystalline bacterial cell surface layers

Abstract Crystalline bacterial cell surface layers (S-layers) are one of the most common outermost cell envelope components of prokaryotic organisms (archaea and bacteria). S-layers are monomolecular arrays composed of a single protein or glycoprotein species and represent the simplest biological membranes developed during evolution. S-layers as the most abundant of prokaryotic cellular proteins are appealing model systems for studying the structure, synthesis, genetics, assembly and function of proteinaceous supramolecular structures. The wealth of information existing on the general principle of S-layers have revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for chemical modifications and binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from a variety of organisms are capable of recrystallizing as closed monolayers onto solid supports (e.g., metals, polymers, silicon wafers) at the air–water interface, on lipid films or onto the surface of liposomes; (iv) functional domains can be incorporated in S-layer proteins by genetic engineering. Thus, S-layer technologies particularly provide new approaches for biotechnology, biomimetics, molecular nanotechnology, nanopatterning of surfaces and formation of ordered arrays of metal clusters or nanoparticles as required for nanoelectronics.

Structural and chemical characterization of S-layers of selected strains ofBacillus stearothermophilus andDesulfotomaculum nigrificans

The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each ofBacillus stearothermophilus andDesulfotomaculum nigrificans were compared. Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent. The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices. In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products. The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic. With the exception of the S-layers of two strains ofB. stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated. Among these strains significant differences in the amount and composition of the glycan portions were found. Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.

Production and characteristics of ultrafiltration membranes with uniform pores from two-dimensional arrays of proteins

Abstract Surface or S-layers, which represent the outermost cell wall component of many eubacteria archaebacteria, were used for the production of isoporous ultrafiltration membranes. S-layers are paracrystalline arrays of protein or glycoprotein subunits that are linked by non-covalent interactions and completely cover the cell surface. The porosity of S-layers ranges from 20 to 50%. So far, channels of 2–6 nm diameter have been identified in S-layer lattices, either by application of high-resolution electron microscopy or by permeability studies. For the production of S-layer ultrafiltration membranes (SUM), the isoporous S-layer material isolated from highly thermophilic eubacteria was deposited on commercially available microfiltration membranes by a pressure-dependent deposition procedure. The active ultrafiltration layer, which was responsible for the rejection characteristics, consisted of coherent S-layer material, whereas the only function of the microfiltration membrane was to provide the mechanical support. In order to improve the stability of the ultrafiltration membranes, the S-layer protein was crosslinked with glutaraldehyde. As expected for an isoporous structure, S-layer ultrafiltration membranes displayed very steep rejection curves, when proteins of progressively increasing molecular weight were taken as test molecules. SUM exhibited a remarkable resistance towards acid, alkali and organic solvents. Depending on the chemical modification procedures performed on the S-layer protein, SUM had different surface net charges. The amount of macromolecules adsorbed to SUM surfaces was strongly influenced by both the solution properties and the membrane surface characteristics.

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

ABSTRACT 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 (rSbpA31-1068). 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 rSpbA31-1068. Labeling of the square S-layer lattice formed by recrystallization of rSbpA31-1068/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.

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

ABSTRACT 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 rSbpA31-1068/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.