PRODUCTION AND FLUORESCENCE-ACTIVATED CELL SORTING OF ESCHERICHIA-COLI EXPRESSING A FUNCTIONAL ANTIBODY FRAGMENT ON THE EXTERNAL SURFACE

We have expressed a single chain Fv (scFv) antibody fragment, consisti ng of the variable heavy and variable light domains from two separate anti-digoxin monoclonal antibodies, on the external surface of Escheri chia coli by fusing it to an Lpp-OmpA hybrid previously shown to direc t heterologous proteins to the cell surface. This scFv fusion was expr essed at a high level and was shown to bind the hapten with high affin ity and specificity. Whole cell ELISAs, fluorescence microscopy, prote ase sensitivity, and flow cytometry all confirmed that the scFv was an chored on the outer membrane and was accessible on the surface. Utiliz ing fluorescence-activated cell sorting, we were able to specifically enrich scFv-producing cells from a 10(5)-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 vitr o production and selection of useful antibody fragments.