Engineering Phage as Tools for Immunosorbent Assays and Enzyme-Substrate Panning

Abstract

Viruses such as filamentous bacteriophage have been widely used in phage display for indentifying novel peptides or proteins that bind specifically to a target of interest. This has been particularly valuable in the biomedical field for drug discovery. The unique structure and characteristics of filamentous phage enabled the genetic modification to be conducted in order to produce various engineered phages which can be used in different applications. In our works, we utilized the engineered phage as a tool for enzyme-substrate biopanning and as a component of a sensing system. The engineered phage with a random XCXPXRX peptide library displayed on its p3 coat protein was used in phage screening for identifying FGE substrates. A pool of XCXPXRX library expressed on the virion surface was modified into aldehyde bearing formylglycine by FGE from M. tuberculosis and covalently immobilized onto an amine bearing surface. The covalently captured phage was eluted by proteolytic cleavage and amplified in E.coli host cells. Our results showed that HCTPRRP was selected and identified as an efficient substrate to undergo FGE modification through the phage screening process, and also as a preferential substrate for proteolytic cleavage. We believe this evolutionary phage screening process can help in identifying specific substrates for various enzymes. Using the concept of natural phage infectivity and a phagemid containing antibiotic resistance genes, we demonstrated a unique phage infectivity based assay in detecting target antigens of interest. The incorporation of a dimer of z-domain from protein A was placed into the p3 gene of the phagemid which enabled the resulting zz-phage to bind to the Fc region of IgG antibodies from several organisms. Through phage infection and selective growth in host E. coli cells by growing in the presence of antibiotic, the detection signal for the presence of antigen could be amplified. The zz-phage acted as a transducer by converting the presence of antigen into visually detectable and measurable colony forming units (C.F.U.). Our phage infectivity assay improved the detection limit by showing a sub-nanomolar detection limit. Although this assay is time consuming, it offers a lower cost than conventional ELISA by replacing the need of enzyme reporter, chromogenic substrate, and spectroscopic equipment with its self regenerating components (phage and bacteria). In summary, engineered phage had shown a great potency to be developed as a tool or new material in different applications. We hope that our phage screening and phage infectivity assay can be carried out in practical use in the future.