Recombinant Protein Expression in Pichia
Dominik Haudenschild April 16, 2001 at UCD
Recombinant -6his fusion protein
- Start from single colonies
- Streak frozen stock on fresh YPD plate containing 100µg/ml Zeocin
- Grow at 28-30¡C until discrete colonies are visible
Generate BioMass
- Pick a single colony
- Grow for 24-36 hours in BMGY media with 5µg/ml Tetracyclene
- Collect yeast by centrifugation at 3000-4000g for 20 minutes
Induce cells for 24-36 hours in BMMY
- Resuspend yeast in 1/10 original volume of BMMY for KM-71H strains
- Resuspend yeast in 2x original volume of BMMY for X-33 strains
Harvest protein in supernatant by centrifuging as before
- Filter (0.2 or 0.4µm) to completely clear supernatant
Purify protein as follows
- Add 1 volume of 2x Binding Buffer to clarified culture supernatant
(2x BB is 100mM Sodium Phosphate pH 7.2, 8% Glycerol, 0.4M NaCl)
- Bind to 3ml of Qiagen Ni-NTA Agarose beads 50% slurry (Cat#????)
pour over column once or twice to allow 6-his to bind
- Wash column with 10ml of 1x Binding Buffer
(1x BB is 50mM Sodium Phosphate pH 7.2, 4% Glycerol, 0.2M NaCl)
- Elute column with 10ml of 1xBB containing 50mM EDTA
- Concentrate to ~500µl on YM-10 centricon then add 1 volume glyceral
- Store in 50% Glycerol at -20¡C
Yield should be about 5mg of DAN from 1 liter culture of Dan-KM71H Clone 8 or 13 (refTLR2p90)