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Applied and Environmental Microbiology, March 2004, p. 1514-1521, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1514-1521.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

Christine Völlenkle,¹ Stefan Weigert,² Nicola Ilk,¹ Eva Egelseer,¹ Viktoria Weber,³ Fritz Loth,⁴ Dieter Falkenhagen,³ Uwe B. Sleytr,¹ and Margit Sára^1*

Center for Ultrastructure Research and Ludwig Boltzmann Institute for Molecular Nanotechnology, University of Natural Resources and Applied Life Sciences,¹ Nano S Biotechnology GmbH, Vienna,² Christian Doppler Laboratory for Specific Adsorption Technologies in Medicine, Centre for Biomedical Technology, Danube University, Krems, Austria,³ Fraunhofer Institute for Applied Polymer Research, Golm, Germany⁴

Received 15 September 2003/ Accepted 25 November 2003

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/mm², whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm² 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.

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* Corresponding author. Mailing address: Zentrum für Ultrastrukturforschung, Universität für Bodenkultur, Gregor Mendelstr. 33, 1180 Vienna, Austria. Phone: 43-1-47 654/2208. Fax: 43-1-478 91 12. E-mail: margit.sara{at}boku.ac.at.

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Applied and Environmental Microbiology, March 2004, p. 1514-1521, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1514-1521.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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