Production and Fluorescence-Activated Cell Sorting of Escherichia coli Expressing a Functional Antibody Fragment on the External Surface

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 90; no. 22; pp. 10444 - 10448
• Main Authors: Francisco, Joseph A., Campbell, Rob, Iverson, Brent L., Georgiou, George
• Format: Journal Article
• Language: English
• Published: Washington, DC National Academy of Sciences of the United States of America 15.11.1993; National Acad Sciences; National Academy of Sciences
• Subjects: Amino Acid Sequence; Antibodies; Bacterial Outer Membrane Proteins - genetics; Bacteriophages; Base Sequence; Biological and medical sciences; Cell membranes; Cell Separation - methods; Cellular biology; Cloning, Molecular - methods; Digoxin - immunology; Escherichia coli; Escherichia coli - isolation & purification; Flow Cytometry - methods; Fluorescence; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Genes, Immunoglobulin; Haptens; Immunoglobulin fragments; Immunoglobulin Variable Region - chemistry; Libraries; Microscopy; Molecular immunology; Molecular Sequence Data; Molecules; Proteins; Recombinant Proteins; Techniques; Recombinant microorganism; Heterologous system; Secretion; Escherichia coli; Selection; Bacteria; Genetic engineering; Recombinant protein; Gene expression; External membrane; Anchoring; Enterobacteriaceae
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.90.22.10444

We have expressed a single chain Fv (scFv) antibody fragment, consisting of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escherichia coli by fusing it to an Lpp-OmpA hybrid previously shown to direct heterologous proteins to the cell surface. This scFv fusion was expressed at a high level and was shown to bind the hapten with high affinity and specificity. Whole cell ELISAs, fluorescence microscopy, protease sensitivity, and flow cytometry all confirmed that the scFv was anchored on the outer membrane and was accessible on the surface. Utilizing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 105-fold excess of control cells in only two steps. The expression of antibody fragments on the surface of E. coli is being evaluated as an attractive method for the in vitro production and selection of useful antibody fragments.