Development of a -lactamase activity assay for detecting ligand-protein interactions using an engineered split intein and -lactamase

Several synthetic compounds bind to proteins of interest and inhibit protein-protein interactions. To develop a detection method for the interactions between the synthetic compounds and the target proteins, we used an engineered split intein derived from Nostoc punctiforme PCC73102 (Npu) DnaE and TEM-1 beta-lactamase as reporter proteins. We constructed synthetic ligands bearing a 6-residue C-terminal peptide from Npu DnaE and Cys-Trp as the C-extein, and target proteins bearing the N-terminal region of the engineered Npu DnaE and residues 24-284 of beta-lactamase. Specific ligand-protein interactions such as phosphopeptide-Src homology domain 2 (SH2) of c-Src and imatinib-quinone reductase 2 (NQO2) increased the protein trans-splicing (PTS) reaction rates and yields. The PTS product showed the enhanced beta-lactamase activity compared with the starting materials. The PTS-based beta-lactamase activity assay was used for the quantitative analysis of the ligand-protein interactions. The signal sequence and 9-residue N-terminal sequence of Escherichia coli (E. coli) lipoprotein (Lpp) and residues 46-159 of outer membrane protein A (OmpA) (LppOmpA) were conjugated with the target proteins bearing split intein and beta-lactamase to display them on live E. coli cell surfaces. PTS on live E. coli surfaces provided enhanced resistance to carbenicillin.