Strains and culture
Sup35 was cloned from 74-D694 [psi -] genomic DNA. Restriction endonuclease digestion sites, Not I and Nco I, were chosen as Sup35 insertion sites for the pET28a expression vector. The primer sequences were as follows: Sup35-Forward, 5’-GTGCCGCGCGGCAGCCATATGTCGGATTCAAACCAAGGC-3’; Sup35- Reverse, 5’-TGGTGGTGCTCGAGTGCGGCCGCTTACTCGGCAATTTTAACAAT -3’. The target pET28a-Sup35 vector was amplified in DH5α (transformed by traditional heat-shock transfer method) and sequenced for the verification of an as-designed sequence. BL21 Rosseta (DE3) cells harboring the pET28a-Sup35 vector were used for the expression and purification of Sup35. The transformants were cultured in LB medium with 50 mg/L kanamycin at 37oC and 220 rpm. When these cells’ OD600 reached 1.0, IPTG was added to the medium with a final concentration of 0.5 mM. The growing condition for these transformants was then switched to 30oC and 160 rpm. After 4 h, the cells were harvested at 4oC and 5000 g for 5 min.
The yeast was of the 74-D694 [psi -] strain. The cells were cultured with YPAD medium (20 mg/mL Ade) for 48 h.
Purification of Sup35 protein
Sup35 induced cells were resuspended in lysis buffer (buffer A, 20 mM Tris, pH 8.0, 1.2 M NaCl, 10 mM PMSF). The bacterial suspension was then sonicated to lyse the cells. The 50 mL lysis mixture from the 1.5 L total culture was centrifuged at 8000 g for 15 min, and the supernatant was filtered by 0.22 μm membranes. All the purification processes were conducted with GE AKTA purifier (pH/c-900, UV-900, P-900, frac-900) at 4oC. An Ni2+-NTA agarose column (His Trap HP, 175248-01) was used for first-stage purification. The column was washed with wash buffer (20 mM Tris, pH 8.0, 20 mM imidazole, 10 mM PMSF, 5% buffer B) for 20 column volumes (flow rate: 1.5 mL/min). The target protein was eluted by 5-50% and 50-100% imidazole gradients with buffer B (20 mM Tris, pH 8.0, 10 mM PMSF, 500 mM imidazole, 1.2 M NaCl). Protein concentration was measured using the BCA protein assay kit (Pierce, product #23227).