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

摘要

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 nanopatterend 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 col.For construction of the S-layer fusion protein,the 3'-end of the sequence encoding the C-terminallyi 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.