Crystalline bacterial cell surface layers (S-layers), S-layer associated cell wall polysaccharides and S-layer associated exoenzymes as building blocks for supramolecular engineering

For novel developments in the field of nanobiotechnology, such as the production of biochips, biosensors, biomimetic immunotherapeutics and vaccines, or artificial viruses, molecules are required, which are able to interlock spontaneously in well-defined manner. Such molecules represent self-assembly systems and they are considered to play an important role in building up supramolecular structures with a dimension > 100 nm. For generating such supramolecular structures, natural self-assembly systems are advantageous, because they have been optimized in the course of evolution and possess the precision of biological molecules. Building up supramolecular structures with a size > 100 nm is meant under bottom-up strategy, while its counterpart, the well known top-down strategy makes use of classical microlithographic techniques. Crystalline bacterial cell surface layer (S-layer) proteins, which represent the outermost cell envelope component of many bacteria and archaea, are a first order self-assembly system. This means that only a single protein or glycoprotein species is required for building up monomolecular protein lattices. Such protein monolayers are only 10 to 15 nm thick and depending on the S-layer protein, they exhibit oblique, square or hexagonal lattice symmetry. In the case of bacteria, the S-layer subunits are linked to one other and to the underlying cell envelope layer by non covalent interactions. Isolated S- layer subunits frequently possess the ability to self-assemble in suspension or to recrystallize into monomolecular protein lattices on solid supports, such as gold substrates, silicon wafers or plastic foils, as well as on lipid films or liposomes. Depending on the physicochemical properties of the underlying material, the S-layer subunits attach with their outer or inner surface. To achieve a uniform orientation, secondary cell wall polymers, which are the natural anchoring molecules for S-layer proteins in gram-positive bacteria, are exploited. For linking them to gold chips, a terminal sulphhydryl group was introduced into the reducing of the polymer chains, which then attached to the gold chips by making use of the auro-thio-chemistry. Atomic force microscopic analysis revealed that the S- layer protein recrystallized into a monomolecular protein lattice on gold chips precoated with secondary cell wall polymer. The advantage of using such modified supports can be seen in the fact that the S-layer subunits will not only bind in pre-determined orientation, but that grain boundaries will be minimized due to the presence of an in vivo-like secondary cell wall polymer layer. In order to functionalize monomolecular protein lattices, there are basically two possibilities, namely functionalization by chemical methods or functionalization by genetic approaches. In the course of functionalization by chemical methods, enzymes, ligands or antibodies are immobilized on S-layer lattices. For producing functional S-layer fusion proteins which retain the ability to self- assemble and carry the secondary cell wall polymer binding region, detailed knowledge on the structure-function- relationship of S-layer proteins was required. In the case of the S-layer proteins investigated so far, the N-terminal part carries the secondary cell wall polymer binding region, while the middle part comprises the self-assembly domain. Therefore, the C-terminal part can be deleted and replaced by functional sequences, such as core streptavidin, the Fc-binding ZZ domain as derived from Protein A, the hypervariable region of camel antibodies or allergens. All S-layer fusion proteins produced up to now have been functional and carried the introduced sequences on the outermost surface of these crystalline arrays. Depending on the type of functional sequence, S- layer fusion proteins shall be exploited for biochip development, or in the case of liposomes, as novel targetting and delivery systems.