Protein expression in yeast

Plasmids carrying the tryptophan nutrition marker gene (TRP+) were then transformed into the yeast parent strain EBY100 (URA+ and TRP−) by electroporation following the BioRad MicroPulser protocol. After 2 days at 30 °C, single yeast colonies can grow on agar plates containing tryptophan dropout medium, e.g., SD-CAA (2% (w/v) glucose, 0.67% (w/v) yeast nitrogen base without amino acids, 0.062% (w/v) Ura/Trp dropout casamino acids, 38 mM Na₂HPO₄, and 62 mM NaH₂PO₄ (pH 6.0)). Two milliliters of SD-CAA minimal medium was then inoculated with a single yeast colony and cultured overnight at 30 °C with shaking at 225 r.p.m. to an OD₆₀₀ of 2.5–5.0. To induce GAL1–10-driven protein expression in yeast, 10⁷ cells were harvested and switched to 2 ml SG-CAA medium (in which glucose was replaced with galactose). After 18 h of induction at 30 °C, a sufficient number of yeast cells per sample were collected by centrifugation at 2500 × g for 3 min, and were then washed and prepared for analysis of protein expression or peptide binding.