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.
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.
Breitwieser
Andreas
A
Center for Ultrastructure Research and Ludwig Boltzmann-Institute for Molecular Nanotechnology, University of Agricultural Sciences, Gregor Mendelstrasse 33, 1180 Vienna, Austria.
Egelseer
Eva M
EM
Moll
Dieter
D
Ilk
Nicola
N
Hotzy
Christoph
C
Bohle
Barbara
B
Ebner
Christof
C
Sleytr
Uwe B
UB
Sára
Margit
M
D000485:Allergens / Q000235:genetics* / Q000276:immunology; D052179:Antigens, Plant; D001426:Bacterial Proteins / Q000235:genetics*; D003460:Crystallization; D001411:Geobacillus stearothermophilus; D015151:Immunoblotting; D007073:Immunoglobulin E / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics* / Q000276:immunology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
D000485:Allergens; D052179:Antigens, Plant; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; C061312:Bet v 1 allergen, Betula; D007073:Immunoglobulin E