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.

TitleAn 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.
Publication TypeJournal Article
Year of Publication2004
AuthorsPleschberger, M., D. Saerens, S. Weigert, U. B. Sleytr, S. Muyldermans, M. Sára, and E. M. Egelseer
JournalBioconjug Chem
Volume15
Issue3
Pagination664-71
Date Published2004 May-Jun
ISSN1043-1802
KeywordsAnimals, Antibodies, Biosensing Techniques, Camels, Cloning, Molecular, Enzyme-Linked Immunosorbent Assay, Epitope Mapping, Gold, Immunization, Male, Nanotechnology, Prostate-Specific Antigen, Recombinant Fusion Proteins, Surface Plasmon Resonance
Abstract

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.

DOI10.1021/bc049964w
Alternate JournalBioconjug. Chem.
PubMed ID15149195