Generation of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.

TitleGeneration of a functional monomolecular protein lattice consisting of an s-layer fusion protein comprising the variable domain of a camel heavy chain antibody.
Publication TypeJournal Article
Year of Publication2003
AuthorsPleschberger, M., A. Neubauer, E. M. Egelseer, S. Weigert, B. Lindner, U. B. Sleytr, S. Muyldermans, and M. Sára
JournalBioconjug Chem
Volume14
Issue2
Pagination440-8
Date Published2003 Mar-Apr
ISSN1043-1802
KeywordsAnimals, Antibodies, Bacillus, Bacteria, Bacterial Proteins, Camels, Cell Wall, Cloning, Molecular, Crystallization, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Escherichia coli, Immunoblotting, Immunohistochemistry, Microcomputers, Microscopy, Atomic Force, Muramidase, Nanotechnology, Peptidoglycan, Polystyrenes, Recombinant Fusion Proteins, Surface Plasmon Resonance
Abstract

Crystalline bacterial cell surface layer (S-layer) proteins are composed of a single protein or glycoprotein species. Isolated S-layer subunits frequently recrystallize into monomolecular protein lattices on various types of solid supports. For generating a functional protein lattice, a chimeric protein was constructed, which comprised the secondary cell wall polymer-binding region and the self-assembly domain of the S-layer protein SbpA from Bacillus sphaericus CCM 2177, and a single variable region of a heavy chain camel antibody (cAb-Lys3) recognizing lysozyme as antigen. 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-Lys3. The functionality of the fused cAb-Lys3 in the S-layer fusion protein was proved by surface plasmon resonance measurements. Dot blot assays revealed that the accessibility of the fused functional sequence for the antigen was independent of the use of soluble or assembled S-layer fusion protein. Recrystallization of the S-layer fusion protein into the square lattice structure was observed on peptidoglycan-containing sacculi of B. sphaericus CCM 2177, on polystyrene or on gold chips precoated with thiolated secondary cell wall polymer, which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Thereby, the fused cAb-Lys3 remained located on the outer S-layer surface and accessible for lysozyme binding. Together with solid supports precoated with secondary cell wall polymers, S-layer fusion proteins comprising rSbpA(31)(-)(1068) and cAbs directed against various antigens shall be exploited for building up monomolecular functional protein lattices as required for applications in nanobiotechnology.

DOI10.1021/bc025603+
Alternate JournalBioconjug. Chem.
PubMed ID12643755