Yeast extract peptone dextrose medium

Phaffia rhodozyma NCYC 874 was obtained from the National Collection of Yeast Culture (UK) and cultured in Yeast Extract-Peptone-Dextrose medium (Sigma Aldrich; Poznań, Poland) for 72 h at 21 °C in a shaker at 160 rpm (LS 500 POL-EKO Aparatura; Wodzisław Śląski, Poland). Cells were afterwards collected by centrifugation at 3200× g for 5 min, 4 °C.
The biomass (5 g) was extracted with 100 mL of acetone (Sigma Aldrich; Poznań, Poland) in a shaker at 160 rpm at 30 °C for 2 h. After pelleting the yeast cells by centrifugation at 3200× g for 5 min, 4 °C the solvent was evaporated in a ventilated incubator completely from the sample at 35 °C in the dark and stored at 4 °C in the dark until use.

In both phage-display libraries, the scFv coding regions are bounded by Hind III and Sal I [52] (link) or Nco I and Not I [53] (link) restriction endonuclease sites. To clone these coding regions into pPICZαC′ expression vector, the phagemid vectors were digested with their respective restriction enzymes (New England BioLabs), the released scFv DNA fragment was isolated (Qiagen, Valencia, CA), and ligated with the similarly digested vector DNA using 1 unit of T4 DNA ligase (New England BioLabs) for every 1 µg of digested vector. The resulting recombinant DNA was used to transform electrocompetent TG1 cells (Lucigen, Middleton, WI) and plated on low-salt LB (1% tryptone, 0.5% yeast extract, 0.5% NaCl, pH 7.5)+25 µg/mL Zeocin™ (Invitrogen, Carlsbad, CA)+15 g/L agar plates and grown overnight at 37°C. The positive clones were digested with Pme I (New England BioLabs) to linearize the DNA and used to transform the X33 strain of P. pastoris, which was grown according to the manufacturer's instructions (Invitrogen). The transformants were grown with varying concentrations of Zeocin™ (0.1 mg/L and 1 mg/L) in YPD medium [Yeast extract Peptone dextrose medium: 1% yeast extract, 2% peptone, 2% dextrose (Sigma, St. Louis, MO)] to select for clones that carried multiple integration events.