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

Abstract

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.

Fig. 1.

Localization of insertion and mutation sites on the three-dimensional structure of TEM-1 β-lactamase. Loops A and C in which insertions were carried out to generate L1B, L3B, and L4 libraries are represented in green. Additional residues mutated in Fer503-1 (T114M, L201P, I287V), Fer503-2 (T114M, N132S, L201Q), and Fer503-5 (T114M) clones are indicated in red. The active site Ser 70 residue is labeled in blue. The model was generated using Molscript V2.1 and Raster 3D V2.5.

Fig. 2.

Characterization of binding properties of (A) β-galactosidase- and (B) ferritin-selected β-lactamases. 10⁷ to 10¹³ phages were incubated per well on microplates coated with β-galactosidase or ferritin. Bound phages were detected with an anti-M13 antibody conjugated to horseradish peroxidase. Readings were carried out at 405 nm every minute for ∼1 h. Readings taken at 60 min (β-galactosidase) or 15 min (ferritin) were plotted as a function of phage concentration. (A) (♦) FdBla; (○) Gal601; (▪) Gal604; (▴) Gal605. (B) (♦) FdBla; (□) Fer503; (▪) Fer503-1; (•) Fer503-2; (○) Fer503-3.

Fig. 3.

Activity modulation of streptavidin- and ferritin-selected β-lactamases as a function of streptavidin or ferritin concentration. Measurements were carried out on 1 mM penicillin G at 20°C. (A) (♦) SVL1-01; (▴) SVL1-03; (▪) SVL3-02; (|qX) FdBla. (B) (▪) Fer503-1; (♦) Fer503-3; (•) purified Fer503-1; (▴) purified Fer503-3; (×) FdBla. (Solid lines) phage–enzymes; (dotted lines) purified enzymes.

Fig. 4.

Fer503-1 and Fer503-3 purification as free enzymes. The purity of Fer503-1 and Fer503-3 was analyzed on a 10% acrylamide gel. Protein bands were revealed by Coomassie blue staining. As control, several different amounts of purified TEM-1 enzymes were loaded. Concentrations of purified enzymes were determined by the bicinconinic assay.

Fig. 5.

Titration of purified Fer503-1 and Fer503-3 enzymes as a function of ferritin concentration by ELISA. First, 5 nM Fer503-1 or Fer503-3 was incubated in Eppendorfs with various concentrations of ferritin in solution. After the equilibrium was reached, the concentration of free β-lactamase in each sample was determined on ferritin-coated 96-well microplates by measuring on nitrocefin the β-lactamase activity that can be immobilized on the plates.

Fig. 6.

Horse spleen ferritin assay. Increasing amounts of purified horse spleen ferritin were incubated on 96-well microplates coated with an anti-horse spleen ferritin polyclonal antibody. Bound ferritin was detected with 50 ng of purified Fer503-3 enzyme per well by measuring on 1 mM nitrocefin the β-lactamase activity immobilized.