title: Selection of TNF-alpha binding affibody molecules using a beta-lactamase protein fragment complementation assay.
abstract: Protein fragment complementation assays (PCAs) based on different reporter proteins have been described as powerful tools for monitoring dynamic protein-protein interactions in living cells. The present study describes the construction of a PCA system based on genetic splitting of TEM-1 beta-lactamase for the selection of proteins specifically interacting in the periplasm of Escherichia coli bacterial cells, and its application for the selection of affibody molecules binding human tumour necrosis factor-alpha (TNF-alpha) from a combinatorial library. Vectors encoding individual members of a naïve 10(9) affibody protein library fused to a C-terminal fragment of the beta-lactamase reporter were distributed via phage infection to a culture of cells harbouring a common construct encoding a fusion protein between a non-membrane anchored version of a human TNF-alpha target and the N-terminal segment of the reporter. An initial binding analysis of 29 library variants derived from surviving colonies using selection plates containing ampicillin and in some cases also the beta-lactamase inhibitor tazobactam, indicated a stringent selection for target binding variants. Subsequent analyses showed that the binding affinities (K(D)) for three selected variants studied in more detail were in the range 14-27 nm. The selectivity in binding to TNF-alpha for these variants was further demonstrated in both a cross-target PCA-based challenge and the specific detection of a low nm concentration of TNF-alpha spiked into a complex cell lysate sample. Further, in a biosensor-based competition assay, the binding to TNF-alpha of three investigated affibody variants could be completely blocked by premixing the target with the therapeutic monoclonal antibody adalimumab (Humira), indicating overlapping epitopes between the two classes of reagents. The data indicate that beta-lactamase PCA is a promising methodology for stringent selection of binders from complex naïve libraries to yield high affinity reagents with selective target binding characteristics.
authors: Division of Molecular Biotechnology, School of Biotechnology, Royal Institute of Technology (KTH), SE-106 91 Stockholm, Sweden.
authors: P-A
authors: PA
authors: Löfdahl
authors: O
authors: O
authors: Nord
authors: L
authors: L
authors: Janzon
authors: P-A
authors: PA
authors: Nygren
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay / Q000379:methods*; D002999:Clone Cells; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000939:Epitopes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1