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).