Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library

Abstract

A nonimmune library of 10(9) human antibody scFv fragments has been cloned and expressed on the surface of yeast, and nanomolar-affinity scFvs routinely obtained by magnetic bead screening and flow-cytometric sorting. The yeast library can be amplified 10(10)-fold without measurable loss of clonal diversity, allowing its effectively indefinite expansion. The expression, stability, and antigen-binding properties of >50 isolated scFv clones were assessed directly on the yeast cell surface by immunofluorescent labeling and flow cytometry, obviating separate subcloning, expression, and purification steps and thereby expediting the isolation of novel affinity reagents. The ability to use multiplex library screening demonstrates the usefulness of this approach for high-throughput antibody isolation for proteomics applications.

Construction and characterization of a pseudo-immune human antibody library using yeast surface display

“…The above results were highly reproducible. Taken together, these results demonstrated that the repertoire of the pseudo-immune scFv library was biased toward the target antigen of DR5, whereas the nonimmune library was not [4].…”

“…Yeast cell labelings for affinity analyses and sortings by flow cytometry were performed as described before [12,13]. Yeast cells were grown in SD-CAA media at 30°C for 20 h to an OD 600 of six, pelleted by centrifugation, and transferred into SG-CAA media and grown at 30°C for 20 h to induce the surface expression human scFv library [4,12,13]. Approximately 10 7 cells from the induced yeast library were incubated with anti-c-myc 9E10 mAb (1:100 dilution) and 1 lM biotinylated antigens at 25°C for 30 min in 0.2 ml PBSB (phosphate-buffered saline, pH 7.4, containing 1 mg/ml BSA).…”

“…The nonimmune human scFv library constructed on the yeast EBY100 cell surface was provided from Prof. Dane Wittrup (Massachusetts Institute of Technology) [4]. The first enrichment of biotinylated antigen-binding yeast was performed by the Miltenyi MACS system following a protocol described previously [4,6].…”

“…The used nonimmune human scFv library was previously constructed from commercial sources of mRNA originated from 29 healthy humans on the yeast cell surface and exhibited about 10 3 -fold larger gene diversity ($1 · 10 9 ) [4], than the pseudo-immune scFv library. The ectodomain of DR5 shows high sequence identity with the ectodomains of DR4 (56%), DcR1 (49%), and DcR2 (57%) [10].…”

“…Phage and ribosome display platform technologies have been extensively used to construct a large scFv or Fab library [2,3]. Recently human scFv and Fab libraries have been generated on the yeast surface, which can be propagated without the growth-mediated clonal diversity loss observed with phage antibody library [4][5][6].…”