Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification
| Autor: | Stefan Weigert, Dieter Falkenhagen, Fritz Loth, Viktoria Weber, Eva M. Egelseer, Nicola Ilk, Margit Sára, Christine Völlenkle, Uwe B. Sleytr |
|---|---|
| Přispěvatelé: | Publica |
| Rok vydání: | 2004 |
| Předmět: |
Recombinant Fusion Proteins
DNA Recombinant In Vitro Techniques Microscopy Atomic Force Applied Microbiology and Biotechnology Immunoglobulin G Autoimmune Diseases Bacterial Proteins Protein A/G Humans Cloning Molecular Binding Sites Membrane Glycoproteins Base Sequence Ecology biology Physiology and Biotechnology Fusion protein Microspheres Protein Structure Tertiary Microscopy Electron Biochemistry IgG binding biology.protein Protein G Crystallization Immunosorbents Protein A S-layer Food Science Biotechnology Binding domain |
| Zdroj: | Applied and Environmental Microbiology. 70:1514-1521 |
| ISSN: | 1098-5336 0099-2240 |
| DOI: | 10.1128/aem.70.3.1514-1521.2004 |
| Popis: | 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 rSbpA 31-1068 /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/mm 2 , whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm 2 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. |
| Databáze: | OpenAIRE |
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