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

Abstract

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-m-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.

Immunoabsorbent nanoparticles based on a tobamovirus displaying protein A

“…The use of a plant-derived immunoabsorbent makes it an especially attractive tool for the purification of plant-made monoclonal antibodies because it can be used in its crude form and does not require extensive processing. The binding capacity of the current CP-protein A (2 g of IgG per 1 g immunoadsorbent or one IgG molecule per 3-5 moieties of protein A) is 200-fold higher than that of the existing protein A carriers (30,37). For industrial applications, future protocols might be developed (based on the same or similar immunoadsorbent polymers) that more fully use the nanoparticle nature of the immunoadsorbent.…”

“…Several potential biotechnological solutions have been described in the literature that propose fusing protein A (or other affinity ligands) to elements capable of polymerizing (bacteriophage capsid proteins [26][27][28]) or binding to structures of high molecular weight (bacterial S-layer proteins [29,30], oleosins recognizing oil bodies [31,32], cellulose-binding domains [33,34], starch-binding domains [35]). So far, only the process based on oleosin fusions (to protein A) has been advanced to the stage of a commercially viable platform (www.sembiosys.com).…”

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent

“…A recent study describes the development of affinity microparticles using recombinant S-layer-expressing tandem copies of the IgGbinding domain from Staphylococcus aureus protein A for use in extracorporeal blood purification (37). These studies carried out with another model system serve to demonstrate, along with the present study, that regularly structured protein lattices can exhibit the functionality of the foreign protein.…”

“…These applications indicate that S-layer proteins offer a novel and effective way to immobilize foreign proteins in the context of a particular biological assay. Although the Bacillus system is complicated by the fact that, unlike with C. crescentus, recombinant proteins have to be expressed in E. coli, the distinct advantage is that recombinant SbpA can be recrystallized in an oriented fashion on a variety of supports precoated with a secondary cell wall polymer (16,24,37). Taken together, the successes obtained with both of these model systems suggest that S-layer fusion proteins can be used in many applications where it is necessary or advantageous to immobilize and/or isolate IgG.…”

“…This is the first published report demonstrating the use of whole Caulobacter cells displaying a fully functional foreign peptide in a research application. In another S-layer model system, B. sphaericus S-layer protein SbpA was first fused to a specific antigen and used as the basis of a dipstick-type immunoassay for the rapid determination of specific IgE in serum (11), and it was used to display tandem IgG-binding sequences from S. aureus protein A to purify autoantibodies from blood (37). These applications indicate that S-layer proteins offer a novel and effective way to immobilize foreign proteins in the context of a particular biological assay.…”