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.
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Zentrum für Ultrastrukturforschung und Ludwig Boltzmann-Institut für Molekulare Nanotechnologie, Universität für Bodenkultur Wien, Gregor-Mendelstr. 33, A-1180, Vienna, Austria.
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D000588:Amines; D000888:Antibodies, Anti-Idiotypic / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001710:Biotin; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D003460:Crystallization; D006801:Humans; D006838:Hydrocarbons; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D008081:Liposomes; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008854:Microscopy, Electron; D011485:Protein Binding
D000588:Amines; D000888:Antibodies, Anti-Idiotypic; D001426:Bacterial Proteins; D002234:Carbodiimides; D003432:Cross-Linking Reagents; D006838:Hydrocarbons; D007074:Immunoglobulin G; D008081:Liposomes; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D001710:Biotin; C013553:hexadecylamine