Engineering Antibody Affinity by Yeast Surface Display

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

“…The oligodeoxynucleotides were synthesized from Bionics Co. (Seoul, Korea). Yeast strain of Saccharomyces cerevisiae EBY100 and the yeast surface display plasmid, pCTCON, were previously described in detail [11][12][13].…”

“…Equal amounts of VH and VL genes were mixed together to generate various combinatorial pools between VH and VL family genes, and then overlap extension PCR was carried out to create scFv gene repertoires. The assembled scFv gene products ($800-850 bp) were purified from 1% agarose gels and concentrated using Pellet Paintä (Novagen) [12]. The scFv antibody gene libraries (10 lg) were mixed with linearized pCTCON (1 lg) by NheI/BamHI digestion, and were transformed into the yeast EBY100 strain by homologous recombination using a Bio-Rad Gene Pulser electroporation apparatus [12,15].…”

“…The assembled scFv gene products ($800-850 bp) were purified from 1% agarose gels and concentrated using Pellet Paintä (Novagen) [12]. The scFv antibody gene libraries (10 lg) were mixed with linearized pCTCON (1 lg) by NheI/BamHI digestion, and were transformed into the yeast EBY100 strain by homologous recombination using a Bio-Rad Gene Pulser electroporation apparatus [12,15]. A total of four transformations were performed in parallel.…”

“…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).…”

“…2), compared with those of reported antibody repertoires expressed in human peripheral blood B cells [20]. The yeast display constructs encoded N-terminal hemagglutinin (8 amino acids) and C-terminal c-myc (10 amino acids) tags flanking the scFv library for quantitative detection of surface-displayed scFvs [11,12]. Significant positive labeling for a C-terminal c-myc epitope tag by flow cytometry demonstrated the expression of the pseudo-immune scFv library on the yeast cell surface (Fig.…”