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Selected Publication:

Weiner, C; Sára, M; Sleytr, UB.
(1994): Novel protein a affinity matrix prepared from two-dimensional protein crystals.
Biotechnol Bioeng. 1994; 43(4):321-330 FullText FullText_BOKU

Abstract:
In this article, we describe a novel type of affinity matrix which was prepared by covalently binding Protein A to crystalline cell surface layers (S-layers) from the gram-positive Clostridium thermohydrosulfuricum L111-69. S-layers were used in the form of cell wall fragments, which were obtained by breaking whole cells by ultrasonification and removing the cell content and the plasma membrane. In these thimble-shaped structures, revealing a size of 1 to 2 mu m, the peptidoglycan-containing laver was covered on both faces with a hexagonally ordered S-layer lattice composed of identical glycoprotein subunits. 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 up to two Protein A molecules were bound per S-layer subunit leading to a dense monomolecular coverage of the immobilization matrix with the ligand. Affinity microparticles were capable of adsorbing IgG from solutions of purified preparations, from artificial lgG-albumin mixtures, and from serum. The amount of IgG bound to affinity microparticles corresponded to the theoretical saturation capacity. Under appropriate conditions, up to 95% of the adsorbed IgG could be eluted again. Affinity microparticles were found to have an extremely low Protein A leakage and a high stability toward mechanical forces. Because pores in the S-layer lattice revealed a size of 4 to 5 nm, immobilization of Protein A and adsorption of IgG was restricted to the outermost surface area. This excludes mass transfer problems usually encountered with affinity matrices prepared from amorphous polymers where more than 90% of the ligands are immobilized in the interior. (C) 1994 John Wiley and Sons, Inc.
Authors BOKU Wien:
Sleytr Uwe B.
BOKU Gendermonitor:


Find related publications in this database (Keywords)
CRYSTALLINE SURFACE LAYERS
PROTEIN A
IMMOBILIZATION
AFFINITY MATRIX
CLOSTRIDIUM THERMOHYDROSULFURICUM


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