A recombinant bacterial cell surface (S-layer)-major birch pollen allergen-fusion protein (rSbsC/Bet v1) maintains the ability to self-assemble into regularly structured monomolecular lattices and the functionality of the allergen

• Published in: Protein engineering Vol. 15; no. 3; pp. 243 - 249
• Main Authors: Breitwieser, Andreas, Egelseer, Eva M, Moll, Dieter, Ilk, Nicola, Hotzy, Christoph, Bohle, Barbara, Ebner, Christof, Sleytr, Uwe B, Sára, Margit
• Format: Journal Article
• Language: English
• Published: England Oxford Publishing Limited (England) 01.03.2002
• Subjects: Allergens - genetics; Allergens - immunology; Antigens, Plant; Bacterial Proteins - genetics; Crystallization; Geobacillus stearothermophilus; Immunoblotting; Immunoglobulin E - immunology; Membrane Glycoproteins - genetics; Plant Proteins - genetics; Plant Proteins - immunology; Protein Engineering; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology
• ISSN: 0269-2139; 1741-0126; 1741-0134
• DOI: 10.1093/protein/15.3.243

The mature crystalline bacterial cell surface (S-layer) protein SbsC of Bacillus stearothermophilus ATCC 12980 comprises amino acids 31-1099 and assembles into an oblique lattice type. As the deletion of up to 179 C-terminal amino acids did not interfere with the self-assembly properties of SbsC, the sequence encoding the major birch pollen allergen (Bet v1) was fused to the sequence encoding the truncated form rSbsC(31-920). The S-layer fusion protein, termed rSbsC/Bet v1, maintained the ability to self-assemble into flat sheets and open-ended cylinders. The presence and the functionality of the fused Bet v1 sequence was proved by blot experiments using BIP1, a monoclonal antibody against Bet v1 and Bet v1-specific IgE-containing serum samples from birch pollen allergic patients. The location and accessibility of the allergen moiety on the outer surface of the S-layer lattice were demonstrated by immunogold labeling of the rSbsC/Bet v1 monolayer, which was obtained by oriented recrystallization of the S-layer fusion protein on native cell wall sacculi. Thereby, the specific interactions between the N-terminal part of SbsC and a distinct type of secondary cell wall polymer were exploited. This is the first S-layer fusion protein described that had retained the specific properties of the S-layer protein moiety in addition to those of the fused functional peptide sequence.