An S-layer heavy chain camel antibody fusion protein for generation of a nanopatterned sensing layer to detect the prostate-specific antigen by surface plasmon resonance technology.
The bacterial cell surface layer (S-layer) protein of Bacillus sphaericus CCM 2177 assembles into a square lattice structure and recognizes a distinct type of secondary cell wall polymer (SCWP) as the proper anchoring structure in the rigid cell wall layer. For generating a nanopatterned sensing layer with high density and well defined distance of the ligand on the outermost surface, an S-layer fusion protein incorporating the sequence of a variable domain of a heavy chain camel antibody directed against prostate-specific antigen (PSA) was constructed, produced, and recrystallized on gold chips precoated with thiolated SCWP. The S-layer protein moiety consisted of the N-terminal part which specifically recognized the SCWP as binding site and the self-assembly domain. The PSA-specific variable domain of the camel heavy chain antibody was selected by several rounds of panning from a phage display library of an immunized dromedary, and was produced by heterologous expression in Escherichia coli. For construction of the S-layer fusion protein, the 3'-end of the sequence encoding the C-terminally truncated form rSbpA(31)(-)(1068) was fused via a short linker to the 5'-end of the sequence encoding cAb-PSA-N7. The S-layer fusion protein had retained the ability to self-assemble into the square lattice structure. According to the selected fusion site in the SbpA sequence, the cAb-PSA-N7 moiety remained located on the outer surface of the protein lattice. After recrystallization of the S-layer fusion protein on gold chips precoated with thiolated SCWP, the monomolecular protein lattice was exploited as sensing layer in surface plasmon resonance biochips to detect PSA.
Pleschberger
Magdalena
M
BMT-Biomolecular Therapeutics GmbH, Brunnerstrasse 59, A-1235 Vienna, Austria.
Saerens
Dirk
D
Weigert
Stefan
S
Sleytr
Uwe B
UB
Muyldermans
Serge
S
Sára
Margit
M
Egelseer
Eva M
EM
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D015374:Biosensing Techniques / Q000379:methods*; D002162:Camelus; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D006046:Gold / Q000737:chemistry; D007114:Immunization; D008297:Male; D036103:Nanotechnology; D017430:Prostate-Specific Antigen / Q000032:analysis / Q000097:blood*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods*
D000906:Antibodies; D011993:Recombinant Fusion Proteins; D006046:Gold; D017430:Prostate-Specific Antigen