Soluble protein expression in S. cerevisiae (GFP-His tag) – 2 litre cultures
Day 1
1. Autoclave 160 ml SD –URA (yeast nitrogen base without amino-acids and CSM –URA
dropout; Formedium) + 2% glucose media in a 500 ml conical flask
2. Transfer 10 ml media to a 50 ml Falcon tube (taped loose cap). Inoculate with a single colony
from a fresh (< 1 week old) –URA plate. Alternatively, inoculate from a glycerol stock (this
may take 2 days to grow up). Shake overnight 280 rpm, 30°C
Day 2
3. Autoclave 4 x 500 ml SD –URA 0.1% glucose media in baffled 1 litre flasks
4. Inoculate 150 ml SD –URA 2% glucose with 1 ml overnight culture. Shake overnight 280 rpm,
30°C
Day 3
5. Pre-warm 4 x 500 ml media to 30°C for 15 min
6. Measure OD600 of a 1:10 dilution of the 150 ml overnight culture. Multiply value by 10, and
calculate volume required to inoculate 500 ml media at an OD600 of 0.12 (e.g. if reading =
0.2, OD = 2, dilution factor = 16.7 and volume required to inoculate 500 ml = 30 ml).
Inoculate 500 ml flasks
7. Prepare 200 ml SD –URA 20% galactose induction material. Heat gently in microwave and
stir until fully dissolved. Sterile-filter (0.22 m) into 4 x 50 ml Falcon tubes (one per flask).
8. When OD600 = 0.5-0.6 (6-8 hours), pour in induction media. Switch heat off and shake for 1620 hours at ambient temperature (~25°C)
Day 4
9. Prepare 50 ml CRB solution (50 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.6 M sorbitol, 1 x
Complete –EDTA protease inhibitor tablet (GE Healthcare)). Chill on ice
10. Prepare BeadBeater chamber by pouring in 0.5 mm acid-washed glass beads to near the top
of the rotor. Seal lid with Parafilm and chill in ice-water
11. Harvest yeast by centrifugation at 4000 x g for 10 min (use 6 x 500 ml Beckman centrifuge
bottles and the JLA10.500 rotor). Discard supernatant (treat with Virkon powder).
Resuspend pellets in 25 ml CRB moving solution from one bottle to the next. Transfer
resuspended cells into BeadBeater chamber.
12. Wash centrifuge bottles with 25 ml CRB, moving solution from one bottle to the next.
Transfer to BeadBeater chamber, filling to ~3 mm below the top.
13. Screw lid on chamber and seal with Parafilm. Place in ice-water outer chamber and screw in
to outer jacket. Place on BeadBeater making sure that the cogs engage. Secure the outer
jacket with the spring clips and fill with ice.
14. Disrupt yeast by beating for 5 x cycles of 1 min on, 1 min off. Top up ice as necessary
15. Disassemble chamber and transfer yeast suspension to 2 x 50 ml Falcon tubes, first using a
10 ml stripette and then using a 1 ml pipette, plunging the tip to the bottom of the beads to
recover as much liquid as possible (optional: wash beads with 25 ml CRB to increase
recovery)
16. Centrifuge suspension at 3000 x g for 5 min to pellet yeast debris and non-disrupted cells.
Transfer supernatant (which should be very cloudy) to 26.4 ml Beckman polycarbonate
centrifuge tubes. Balance tubes by mass
17. Centrifuge at 33000 rpm (100000 x g) for 1 h in the Optima ultracentrifuge using the 50.2 Ti
rotor to pellet membranes. Transfer supernatant to 2 x 50 ml Falcon tubes.
18. Add 25 ml wash buffer (10 mM Tris-HCl pH 7.9, 150 mM NaCl, 25 mM imidazole, 1 mM
TCEP) and 50 l of a 1:1 slurry of washed Ni-sepharose beads (GE Healthcare; use a pipette
tip with the end cut off) to each tube. Rotate at room temperature for 1 h
19. Centrifuge at 1000 x g for 2 min to pellet beads. Allow beads to settle at the bottom of the
tubes by standing them in a tube rack on the bench for 10 min
20. Remove supernatant carefully (take a 1 l sample for SDS-PAGE (FT))
21. Transfer and pool beads in an Eppendorf tube. Centrifuge 5000 rpm for 10 s to pellet beads.
Discard supernatant. Beads should be green.
22. Wash beads by adding 0.5 ml wash buffer, mixing and centrifuging at 5000 rpm for 10 s.
Save a 10 ml sample for SDS-PAGE (W1)
23. Repeat step 22 5 times (6 washes in total). Save a 10 ml sample of wash 6 for SDS-PAGE (W6)
24. Elute protein by adding 0.5 ml elution buffer (10 mM Tris-HCl pH 7.9, 150 mM NaCl, 500 mM
imidazole, 1 mM TCEP), mixing and centrifuging at 5000 rpm for 10 s. Take 2 x 10 l samples
for SDS-PAGE (E1nb, E1b). The elution should be green
25. Repeat step 24 (E2) Remaining beads should be blue.
26. Store elution fractions at 4°C (short-term) or at -80°C in 20% glycerol (long-term).
27. Make a 1 g sample of BSA for yield estimation on SDS-PAGE
28. Add 10 l 2x sample buffer (YSB recipe) to each sample. Boil 1x E1 and 1 x E2 sample at 96°C
for 2 min (E1b and E2b). Do not boil the other samples
29. Separate FT, W1, W6, E1nb, E1b E2nb, E2b and BSA on a home-made 12% acrylamide gel
30. Image GFP in-gel fluorescence using the Chemidoc XRS. Stain with InstantBlue for at least 15
minutes and destain with 3x 5 min washes of water.