cNGF-Tanezumab/mAb#1 enrichment ratios sort 2

Activity

Enrichment

unitless

For each sort 200,000 cells were collected using a diagonal gate set to collect the top 2–3% of the displaying population , Yeast Surface Display , Deep Mutational Scanning

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Yeast surface display expression: Cellular fluorescence was measured using a BD Accuri C6 flow cytometer. Yeast cells displaying cNGF variants were detected using anti-cymc-FITC (Miltenyi Biotec, San Diego, CA) and an anti-FLAG tag alexa fluor 647-conjugated antibody (R&D System, Minneapolis, MN). Binding to biotinylated mAbs was detected using streptavidin-R- phycoerythrin conjugate (Thermo Fisher).

Screening: 1E7 cells were grown from freezer stocks in 1 ml of SDCAA for 6 hr at 30°C and re-inoculated at OD600 = 1.0 in 1 ml of SGCAA at 18°C for 16 hrs. 2 × 107 yeast libraries were labeled with either biotinylated tanezumab or mAb #1 at 5 nM for 30 mins at room temperature in PBS-BSA. After centrifugation and washing, cells were secondarily labeled with 60 μl of anti-cymc-FITC and 50 μl of streptavidin-R- phycoerythrin conjugate in 1.89 ml of PBS-BSA for 10 mins at 4°C. Sorting was done on a BD Influx Cell Sorter at the Michigan State University Flow facility. For each sort 200,000 cells were collected (approx. 100-fold the theoretical diversity at the amino acid level) using a diagonal gate set to collect the top 2–3% of the displaying population. Collected cells from each population were recovered at 30°C for 30 hrs in 10 ml of SDCAA and 100 μl of penicillin-streptomycin, washed, and then stored in 1 ml of yeast storage buffer at a concentration of 4E7 cells per ml at −80°C.