Frozen ez kit

Genes of variable heavy (V_H) and variable light (V_L) domains of antibody 3A2 (Nam, et. al. 2016 (link)) were amplified to assemble 3A2 scFv (V_H-SGGSGGGGSGSGS-V_L) by overlapping PCR. Error-prone PCR of 3A2 scFv gene was performed by using Taq DNA polymerase with 120 μM dATP, 100 μM dCTP, 360 μM dGTP, 2.5 mM dTTP, 5 μg/mL BSA, 3.28 mM MgCl₂ and 0.5 mM MnCl₂. The generated mutagenesis product was cloned into the yeast display plasmid pCTcon2 (Feldhaus et al., 2003 ) by transforming 5 μg ligated DNA into E. coli competent cells. 100 μg of library plasmid DNA was used to chemically transform S. cerevisiae EBY100 competent cells prepared by Frozen-EZ kit (Zymo). Transformants were selected on SD/-Trp/-Ura (Sunrise Science) agar plates, then collected and stored at −80 °C. Library quality and the mutation rate was analyzed by DNA sequencing of randomly picked clones. For surface display, 5×10⁹ library cells were cultured on SD/-Trp/-Ura/penicillin-streptomycin agar plates at 30 °C for 48 h. 30 OD₆₀₀ of cultured cells were inoculated to 600 mL SD/-Trp/-Ura for incubation at 30 °C, 250 rpm for 12 h. Cells were collected by centrifugation at 6,000 × g for 2 min, and 8 OD₆₀₀ cells were further cultured for scFv expression in 20 mL YNB (yeast nitrogen base)/-Trp/-Ura supplemented with 5 mL 20% galactose at room temperature 250 rpm for 48 h.