Molecular Characterization of the S-Layer Gene,sbpA, ofBacillus sphaericusCCM 2177 and Production of a Functional S-Layer Fusion Protein with the Ability To Recrystallize in a Defined Orientation while Presenting the Fused Allergen

Abstract

Crystalline bacterial cell surface layers (S-layers) represent the outermost cell envelope component of many bacteria and archaea (35,37,38). S-layers are composed of identical protein or glycoprotein subunits, and they completely cover the cell surface during all stages of bacterial growth and division. The S-layer subunits assemble into either oblique, square, or hexagonal lattices. In the case of Bacillaceae, the N-terminal part is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer (SCWP) to the rigid cell wall layer (2,5,21,25,34,35).S-layers are unique biomaterials with properties most relevant for applications in molecular nanotechnology, nanobiotechnology, and biomimetics (38). Many S-layer proteins recrystallize into regularly structured monolayers on solid supports, such as silicon wafers, gold chips, and silanized glass or plastic materials, as well as on Langmuir lipid films, on liposomes (20,23), and at the air-water interface. Pores passing through S-layer lattices are of identical size and morphology, and functional groups show a regular distribution and high density. For production of S-layer-based biosensors (38), affinity microparticles (45), and solid-phase immunoassays (3,4,39), functional groups in the S-layer lattice were exploited as covalent binding sites for biologically active macromolecules, such as enzymes, antibodies, or ligands. As alternatives to the existing technology, namely, immobilization by chemical methods, genetic approaches are particularly attractive for incorporation of functional peptide sequences into S-layer proteins, which must be done at positions that do not interfere with their self-assembly properties and their interaction with SCWP.

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

“…As shown in a previous study, the deletion of 200 C-terminal amino acids led to a significant increase in the accessibility of the C terminus, while retain- ing the functionality of the S-layer protein moiety (12). The Slayer fusion protein rSbpA /ZZ could self-assemble in suspension and recrystallize on peptidoglycan-containing sacculi and on solid supports precoated with thiolated SCWP.…”

“…The nucleotide sequence encoding the S-layer protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCRbased technique (12). The protein precursor includes a 30-amino-acid-long typical gram-positive signal peptide and consists of a total of 1,268 amino acids.…”

“…Studies of the structure-function relationship of SbpA re-vealed that 200 C-terminal amino acids can be deleted without interfering with the self-assembly properties or the formation of the square lattice structure (12). Furthermore, amino acid position 1,068 was found to be located on the outer S-layer surface and was therefore exploited as a fusion site for the production of chimeric S-layer proteins.…”

“…Furthermore, amino acid position 1,068 was found to be located on the outer S-layer surface and was therefore exploited as a fusion site for the production of chimeric S-layer proteins. So far, S-layer fusion proteins have been constructed that comprised the sequence of the major birch pollen allergen (12) or the sequence of a single variable region of a heavy chain camel antibody directed against lysozyme (24). In the present study, an S-layer fusion protein comprising the sequence of the Z-domain, a synthetic analogue of the immunoglobulin G (IgG)-binding B-domain of protein A of Staphylococcus aureus, has been constructed.…”

High-Affinity Interaction between the S-Layer Protein SbsC and the Secondary Cell Wall Polymer ofGeobacillus stearothermophilusATCC 12980 Determined by Surface Plasmon Resonance Technology

“…Interestingly, in SbpA, as well as in SbsC, the functional SCWP-binding domain comprises the N-terminal amino acid residues 31 to 270 and has been found to be organized mainly as ␣-helices (10,12,18). To conclude, the highly specific lectin-type binding between S-layer proteins and SCWPs is an important mechanism for generating and maintaining a dynamic protein crystal on a bacterial cell surface during all stages of cell growth and division.…”