For VLR surface display assays, EBY100 yeast were grown in SD-CAA medium [dextrose (20.0 g/liter), yeast nitrogen base (6.7 g/liter), casamino acids (5.0 g/liter), Na2HPO4·7 H2O (10.19 g/liter), and NaH2HPO4·H2O (8.56 g/liter)] as previously described and induced when the culture reached an optical density at 600 nm (OD600nm) between 0.8 and 0.9 using SG-CAA medium (21). Yeast was induced for 48 hours at room temperature before VLR or VLR-intein was harvested. For VLR secretion, YVH10 yeast transformed with pRS316–VLR-intein was grown in SD-2XSCAA + Trp [dextrose (20 g/liter), yeast nitrogenous base (6.7 g/liter), Na2HPO4·7H2O (10.19 g/liter), NaH2HPO4·H2O (8.56 g/liter), Arg (190 mg/liter), Met (108 mg/liter), Tyr (52 mg/liter), Ile (290 mg/liter), Lys (440 mg/liter), Phe (200 mg/liter), Glu (1260 mg/liter), Asp (400 mg/liter), Val (480 mg/liter), Thr (220 mg/liter), Gly (130 mg/liter), and Trp (40 mg/liter), lacking leucine and uracil]. Cultures were initiated, and then, cell density reset the following day to an OD600nm = 0.1 and grown for 72 hours at 30°C. Yeast were induced by replacing the medium with an equivalent volume of SG-2XSCAA + Trp [galactose replacing dextrose (20 g/liter)] containing 0.1% (w/v) BSA and culturing the cells for 72 hours at 20°C. Yeast supernants were harvested, filtered through 0.22-μm PES (polyethersulfone) membranes, and dialyzed against tris-buffered saline (TBS) [25 mM tris, 300 mM NaCl, 2 mM KCl (pH 7.9)]. Imidazole (5 mM) plus 200 ml of the dialyzed supernant was gently mixed overnight at 4°C with 1 ml of cobalt HisPur resin (Thermo Fisher Scientific). Beads were collected, washed with TBS plus 10 mM imidazole, and eluted with TBS plus 250 mM imidazole. Elutants were then buffer-exchanged into 50 mM HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] (pH 7.2) (removing the imidazole and neutralizing pH) using 10 K MWCO (10 kDa molecular weight cut-off) filters. Of note, regenerated cellulose membranes contain high amounts of thiols that induce nondesirable intein cleavage during isolation. We recommend using a PES membrane available through Pierce and Pall for buffer exchanges with secreted VLR-intein fusions before expressed protein ligation (EPL).

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