title: Molecular characterization of the S-layer gene, sbpA, of Bacillus sphaericus CCM 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: The nucleotide sequence encoding the crystalline bacterial cell surface (S-layer) protein SbpA of Bacillus sphaericus CCM 2177 was determined by a PCR-based technique using four overlapping fragments. The entire sbpA sequence indicated one open reading frame of 3,804 bp encoding a protein of 1,268 amino acids with a theoretical molecular mass of 132,062 Da and a calculated isoelectric point of 4.69. The N-terminal part of SbpA, which is involved in anchoring the S-layer subunits via a distinct type of secondary cell wall polymer to the rigid cell wall layer, comprises three S-layer-homologous motifs. For screening of amino acid positions located on the outer surface of the square S-layer lattice, the sequence encoding Strep-tag I, showing affinity to streptavidin, was linked to the 5' end of the sequence encoding the recombinant S-layer protein (rSbpA) or a C-terminally truncated form (rSbpA(31-1068)). The deletion of 200 C-terminal amino acids did not interfere with the self-assembly properties of the S-layer protein but significantly increased the accessibility of Strep-tag I. Thus, the sequence encoding the major birch pollen allergen (Bet v1) was fused via a short linker to the sequence encoding the C-terminally truncated form rSpbA(31-1068). Labeling of the square S-layer lattice formed by recrystallization of rSbpA(31-1068)/Bet v1 on peptidoglycan-containing sacculi with a Bet v1-specific monoclonal mouse antibody demonstrated the functionality of the fused protein sequence and its location on the outer surface of the S-layer lattice. The specific interactions between the N-terminal part of SbpA and the secondary cell wall polymer will be exploited for an oriented binding of the S-layer fusion protein on solid supports to generate regularly structured functional protein lattices.
authors.affiliation: Center for Ultrastructure Research, Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, Gregor Mendelstrasse 33, 1180 Vienna, Austria.
authors.forename: Nicola
authors.initials: N
authors.lastname: Ilk
authors.forename: Christine
authors.initials: C
authors.lastname: Völlenkle
authors.forename: Eva M
authors.initials: EM
authors.lastname: Egelseer
authors.forename: Andreas
authors.initials: A
authors.lastname: Breitwieser
authors.forename: Uwe B
authors.initials: UB
authors.lastname: Sleytr
authors.forename: Margit
authors.initials: M
authors.lastname: Sára
mesh_terms: D001407:Bacillus / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D003460:Crystallization; D004926:Escherichia coli; D015870:Gene Expression; D007150:Immunohistochemistry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics*; D009004:Monosaccharide Transport Proteins / Q000235:genetics* / Q000302:isolation & purification; D010446:Peptide Fragments / Q000235:genetics; D011107:Polylysine / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; C120455:SbpA protein, bacteria; D011107:Polylysine; D003300:Copper