Screening combinatorial polypeptide libraries using bacterial surface display and fluorescence -activated cell sorting

A quantitative system for screening combinatorial polypeptide libraries was developed using display on the surface of E. coli and fluorescence-activated cell sorting (FACS). This system enabled the identification of important affinity determinants in the binding pocket of an antibody, and allowed the isolation of antibodies with improved affinity for digoxin and digoxigenin. The surface display system enabled quantitative selection of antibodies based on the kinetic parameters of the antibody-antigen complex. The E. coli surface display system was then redesigned to introduce several characteristics desirable for library screening applications. Modifications were made to: (a) enable regulated production of the displayed fusion protein which prevented the rapid outgrowth of clones producing non-functional antibodies in mutant libraries (b) confer rapid growth compatible with 24 hr screening cycles, shortening the time required for affinity maturation by half The tightly-regulated display system was employed to enhance the affinity of the scFv(dig) antibody towards digoxigenin, a commonly used molecular tag for various molecular biology applications. Error-prone PCR was used to introduce random mutations into the wild-type scFv, creating libraries containing greater than 106 members. After five rounds of kinetic selection using FACS, mutant antibodies with improved affinity and expression levels were isolated. In addition to demonstrating the usefulness of this new display technology, our approach using flow cytometry to characterize large mutant populations has provided new insight into an antibody's tolerance to amino acid substitutions.