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TEM-1 ß-lactamase as a scaffold for protein recognition and assay

Laboratoire de Biochimie Physique et des Biopolyméres, Institut des Sciences de la Vie, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium

Reprint requests to: Dr. Daniel Legendre, Laboratoire de Biochimie Physique et des Biopolyméres, Institut des Sciences de la Vie, Université Catholique de Louvain, Place Pasteur, 1-1B, 1348 Louvain-la-Neuve, Belgium; e-mail: legendre{at}bioc.ucl.ac.be; fax: 3210472820.

A large number of different proteins or protein domains have been investigated as possible scaffolds to engineer antibody-like molecules. We have previously shown that the TEM-1 ß-lactamase can accommodate insertions of random sequences in two loops surrounding its active site without compromising its activity. From the libraries that were generated, active enzymes binding with high affinities to monoclonal antibodies raised against prostate-specific antigen, a protein unrelated to ß-lactamase, could be isolated. Antibody binding was shown to affect markedly the enzyme activity. As a consequence, these enzymes have the potential to be used as signaling molecules in direct or competitive homogeneous immunoassay. Preliminary results showed that ß-lactamase clones binding to streptavidin could also be isolated, indicating that some enzymes in the libraries have the ability to recognize proteins other than antibodies. In this paper, we show that, in addition to ß-lactamases binding to streptavidin, ß-lactamase clones binding to horse spleen ferritin and ß-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining ß-lactamases with affinities comprised between 10 and 20 nM (K_d) for the protein. Contrary to what was observed for ß-lactamases issued from selections on antibodies, enzyme complexation induced only a modest effect on enzyme activity, in the three cases studied. This kind of enzyme could prove useful in replacement of enzyme-conjugated antibodies in enzyme-linked immunosorbant assays (ELISA) or in other applications that use antibodies conjugated to an enzyme.

Keywords: ß-Lactamase; phage display; protein scaffold; protein assay; combinatorial libraries

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