TEM-1 beta-lactamase as a scaffold for protein recognition and assay.
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 beta-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 beta-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 beta-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 beta-lactamases binding to streptavidin, beta-lactamase clones binding to horse spleen ferritin and beta-galactosidase could be isolated. Affinity maturation of a clone binding to ferritin allowed obtaining beta-lactamases with affinities comprised between 10 and 20 nM (Kd) for the protein. Contrary to what was observed for beta-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.
Legendre
Daniel
D
Laboratoire de Biochimie Physique et des Biopolyméres, Institut des Sciences de la Vie, Université Catholique de Louvain, 1348 Louvain-la-Neuve, Belgium. legendre@bioc.ucl.ac.be
Vucic
Bénédicte
B
Hougardy
Vincent
V
Girboux
Anne-Lise
AL
Henrioul
Christophe
C
Van Haute
Julien
J
Soumillion
Patrice
P
Fastrez
Jacques
J
D000818:Animals; D019020:Directed Molecular Evolution; D005293:Ferritins / Q000378:metabolism; D014644:Genetic Variation; D006801:Humans; D007700:Kinetics; D009154:Mutation; D019151:Peptide Library; D011485:Protein Binding; D019809:Streptavidin / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D019151:Peptide Library; D005293:Ferritins; D019809:Streptavidin; D001616:beta-Galactosidase; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1