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 the isolation and in vitro evolution of these biologically important molecules. However, the general usefulness of this technology is still limited by some technical difficulties. One of the most debilitating obstacles to the widespread 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 difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned. Here, we report the construction of a novel phage-display system designed to address these problems. In our system a single-chain Fv (scFv) is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) derived from the Clostridium thermocellum cellulosome. The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof. We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires.
Berdichevsky
Y
Y
Department of Molecular Microbiology, The George S. Wise Faculty of Life Sciences, Green Building, Room 202, Tel-Aviv University, Ramat Aviv 69978, Israel.
Ben-Zeev
E
E
Lamed
R
R
Benhar
I
I
D000818:Animals; D000906:Antibodies / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D003001:Cloning, Molecular; D003013:Clostridium / Q000235:genetics* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000906:Antibodies; D001426:Bacterial Proteins; D017931:DNA Primers; D007128:Immunoglobulin Fragments; D019151:Peptide Library; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv; D002482:Cellulose