PMID:20423457
| Citation |
Mazor, Y, Van Blarcom, T, Carroll, S and Georgiou, G (2010) Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening. FEBS J. 277:2291-303 |
|---|---|
| Abstract |
Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process. |
| Links |
PubMed Online version:10.1111/j.1742-4658.2010.07645.x |
| Keywords |
Antibody Affinity/immunology; Antibody Specificity/immunology; Antigens, Bacterial/immunology; Bacterial Toxins/immunology; Bacteriophages/chemistry; Bacteriophages/genetics; Bacteriophages/metabolism; Capsid Proteins/genetics; Digoxin/immunology; Enzyme-Linked Immunosorbent Assay; Escherichia coli/genetics; Escherichia coli/metabolism; Escherichia coli/virology; Flow Cytometry; Genetic Vectors/genetics; Immunoglobulin G/biosynthesis; Immunoglobulin G/genetics; Immunoglobulin G/immunology; Immunoglobulin G/isolation & purification; Kinetics; Peptide Library; Recombinant Proteins/biosynthesis; Recombinant Proteins/genetics; Recombinant Proteins/immunology; Recombinant Proteins/isolation & purification; Spheroplasts/chemistry; Spheroplasts/metabolism |
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