Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering

Phage display of antibody fragments has proved to be a powerful tool for th e isolation and in vitro evolution of these biologically important molecule s. However, the general usefulness of this technology is still limited by s ome technical difficulties. One of the most debilitating obstacles to the w idespread application of the technology is the accumulation of "insert loss " clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted. Another difficult y arises when phage technology is applied for cloning hybridoma-derived ant ibody genes, where myeloma derived light chains, irrelevant to the hybridom a's antibody specificity may be fortuitously cloned. Here, we report the co nstruction of a novel phage-display system designed to address these proble ms. In our system a single-chain Fv (scFv) is expressed as an in-frame fusi on protein with a cellulose-binding domain (CBD) derived from the Clostridi um thermocellum cellulosome. The CBD domain serves as an affinity tag allow ing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produ ced thereof. We demonstrate the utility of our system in solving the techni cal difficulties described above, and in speeding up the process of scFv is olation from combinatorial antibody repertoires. (C) 1999 Elsevier Science B.V. All rights reserved.