tailieunhanh - Báo cáo khoa học: Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening

Phage display of antibody libraries is a powerful tool for antibody discov-ery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab¢) recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluores-cence-activated cell sorting (FACS) [Mazor Yet al. | ỊFEBS Journal Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening Yariv Mazor1 2 Thomas Van Blarcom1 2 Sean Carroll1 2 and George Georgiou1 2 1 Institute for Cellular and Molecular Biology University of Texas at Austin TX USA 2 Department of ChemicalEngineering University of Texas at Austin TX USA Keywords fluorescence-activated cell sorting FACS full-length IgG fUSE5-ZZ phage protective antigen PA spheroplasts Correspondence G. Georgiou Department of Chemical Engineering University of Texas at Austin Austin TX 78712 USA Fax 1 512 471 7963 Tel 1 512 471 6975 E-mail gg@ Received 7 February 2009 Revised 4 March 2010 accepted 9 March 2010 doi 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 108 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 .