Surface Layers

In microorganisms of almost every taxonomic group of walled eubacteria and archaeobacteria [136, 137] the outer cell wall has a nanoporous crystalline surface layer (S-layer). This surface layer is composed of a single protein or glyco-protein species with molecular weights ranging from 40,000 to 200,000 and can exhibit either oblique, square, or hexagonal lattice symmetry [138-140]. See Figure 57.

An important feature of these nanoporous structures is their repetitive physicochemical properties. Pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes. Functional groups on the surface and attached in the pores may be well aligned and are accessible for specific molecules in a very precise fashion. S-layer proteins also reveal the ability to self-assemble into two-dimensional crystalline arrays in suspension, on solid supports, at the air/water interface, and on lipid films [141].

Modification of S-layers [145] would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein. S-layer proteins can also be produced in large amounts by continuous cultivation of S-layer-carrying organisms. S-layer proteins can be considered biopolymeric membranes with properties ideally tailored by nature for many biotechnolog-ical applications [146], as matrices for the immobilization of a variety of materials including biologically active macro-molecules [147, 148] and for functionalization of synthetic materials. The regular pore properties of S-layers are in contrast with the amorphous pore structure of phase inversion membranes and may therefore be used to produce artificial biomembranes showing excellent molecular mass cutoff separation capabilities [149]. See Figure 58.

One of the most common organisms used for producing S-layer-carrying cell wall fragments and S-layer protein for biotechnological applications is Bacillus stearothermophilus

Figure 57. SEM photograph of a liposome with a nanoporous cell envelope (surface layer). Right: a reconstruction of an S-layer structure (bar = 10 nm). S-layers are formed by self-assembly and are made of protein and glycoproteins (MW 40,000-200,000) with an oblique, square, or hexagonal 2D crystal structure with typical lattice constants between 5 and 30 nm and a thickness of 5-10 nm. Courtesy of Nanosearch membrane GmbH.

Figure 56. Schematic illustration of the assembly of a pore channel with («-hemolysin) peptide oligomers in a (diphy-tanoylphosphatidylcholine) lipid bilayer. Intermediate structures of the assembly process are not considered.

Figure 57. SEM photograph of a liposome with a nanoporous cell envelope (surface layer). Right: a reconstruction of an S-layer structure (bar = 10 nm). S-layers are formed by self-assembly and are made of protein and glycoproteins (MW 40,000-200,000) with an oblique, square, or hexagonal 2D crystal structure with typical lattice constants between 5 and 30 nm and a thickness of 5-10 nm. Courtesy of Nanosearch membrane GmbH.

glass pipette antibody lipid bilayer lipid bilayer water microsieve water microsieve glass pipette

lipid bilayer

Figure 58. (a) Schematic illustration of a "black" lipid membrane generated by the method of Montal and Mueller on a micro/nanosieve. Originally this method involved the generation of a lipid bilayer over a septum with an orifice of 40-800 micrometers [142,143]. For this purpose either a small drop of lipid dissolve in alkane is placed on the opening of the septum [144] or the membrane is formed from two lipid monolayers at an air/water interface by the position of the hydrocarbon chains through an aperture made in a hydrophilic partition which separates the two monolayers. (b) Schematic illustration of a lipid bilayer, generated on the tip of a patch clamp pipette.

PV72, for which a synthetic growth medium has been developed [150, 151]. S-layers have also been used for the production of S-layer based ultrafiltration membranes (SUMs) [152]. SUMs are produced by S-layer self-assembly or by depositing S-layer-carrying cell wall fragments on normal microfiltration membranes. Adsorption studies and contact angle measurements have confirmed that these SUMs are net negatively charged and basically hydrophilic [153].

S-layers may also be used as matrices for covalent binding of biologically active macromolecules, such as enzymes (invertase, glucose oxidase, glucuronidase, L-glucosidase, naringinase, peroxidase), ligands (protein A, streptavidin), or mono- and polyclonal antibodies. S-layer-carrying cell wall fragments with immobilized protein A could be applied as escort particles in affinity cross-flow filtration for isolation and purification of human IgG from serum or of monoclonal antibodies from hybridoma cell culture super-natants [154, 155]. SUMs with immobilized monoclonal antibodies were also used as reaction zones for dipstick-style immunoassays [156]. For this purpose, human IgG was either directly coupled to the carbodiimide-activated car-boxyl groups of the S-layer protein, or it was adsorbed onto a SUM with covalently bound protein A. Alternatively, human IgG was biotinylated and bound to a SUM onto which strep-tavidin was immobilized in a monomolecular layer [157]. See Figure 59.

The experimental use of crystalline bacterial surface layer proteins (S-layers) as combined carrier/adjuvants for vaccination and immunotherapy has since 1987 progressed in three areas of application: immunotherapy of cancers, antibacterial vaccines, and antiallergic immunotherapy. See Figure 60.

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