Editing Pichia EXpression (section)

== Scale up protein expression in PichiaPink stains  ==

1. Using a single colony, inoculate 25 mL of BMGY in a 250 mL baffled flask. Grow at 24–30°C in a shaking incubator (250–300 rpm) until culture reaches an OD600 of 2–6 (approximately 2–3 days). 

2. Use this 25 mL culture to inoculate 1 liter of BMGY in a 3 or 4 liter baffled flask and grow at 24–30°C with vigorous shaking (250–300 rpm) until the culture reaches log phase growth (OD600 = 2–6). 

3. Harvest the cells by centrifuging in sterile centrifuge bottles at 1,500–3,000 × g for 5 minutes at room temperature. To induce expression, decant the supernatant and resuspend the cell pellet in 200 mL of BMMY medium. 

Note: The resuspension step above concentrates the culture 5-fold, thereby reducing the need for concentrating the secreted proteins if the expression level is not particularly high. 

4. Aliquot the culture between two 1 liter baffled flasks. Cover the flasks with PureLink Air Porous Tape, 2 layers of sterile gauze, or<br> cheesecloth and return to incubator. Continue to grow at 24–30°C with shaking. 

5. Add 100% methanol to 0.5% every 24 hours until the optimal time of induction is reached as determined from the time course study. 

6. Harvest cells by centrifuging at 1,500–3,000 × g for 5 minutes at room temperature. 

7. For intracellular expression, decant the supernatant. You can process the cells immediately or store at –80°C until ready for use.<br>8. For secreted expression, save the supernatant, chill to 4°C, and concentrate if desired (see below). Proceed directly to protein purification,<br> or store the supernatant at –80°C until ready to process further.<br><br> 

'''Protocol by Day'''<br> <br> 

'''Day 1-3 or 1-4''': 

streak the transformed yeast stock on an YPDa plate to grow single colonies at 29C. 

<br> 

'''Day 4 or 5'''<br>Starter culture: Culture a single colony in 15 ml BMGY medium in a pathogenic free 125-ml baffled flask with pore cap at 29C at 250rpm shaking until culture reach an OD600 of 2-6 (approx 24 h or 2-3 days to OD600 of 4-5)<br> 

'''Day 6-7'''<br>Use the 15 ml of the starter culture to inoculate 500 ml BMGY in 2-liter pathogenic free baffled flask with pore caps to a final OD600 of 0.1-0.2. Grow at 29C with vigorous shaking (250-300 rpm) until the culture reaches log phase of OD600 2-6. <br> 

'''Day 8''' (Induction Day1)<br>Harvest cells by centrifuging in sterile bottles (Beckman coulter) at 2,500 x g for 5 min at room temperature. Decant the supernatant and resuspend the cells in 100 ml BMMY (0.5% methanol) in a pathogenic free 500-ml flask with pore. Culture the cell at 29 oC and 250rpm shaking. (take 100 ul sample at zero time of induction for SDS-PAGE analysis) 

<br>'''Day 9 '''(Induction Day 2): 

Add 0.6 ml 100% methanol (sterile using 0.22 um on a sterile 250-ml plastic filtration device) to the 100 ml culture in BMMY and continue the culture. (take 100 ul 24h sample for analysis)<br>'''Day 10''' (Induction Day 3, 48 h) 

<br>Harvest the cells by centrifuging in sterile bottles&nbsp; at 2,500 x g for 10 min at 4 oC. Collect the clear supernatant and frozen in liquid nitrogen, store at -80 oC. (take 100 ul supernatant for the analysis). 

'''Media''' for protein expression under low endotoxin condition<br>BMGY or BMMY (Use endotoxin free water or ultrapure water to prepare): 

<br>'''YP solution''': 

700 ml containing 

10 g yeast extract, 

20 g peptone 

100 mM potassium phosphate (autoclaved). 

<br>Add the following sterile solutions to the 700 ml YP:<br>100 ml of 10X YNB<br>100 ml of 10x glycerol stock (10%) <br>20 ml of 50X Biotin (0.02% diluted from 100 mM (2.4% biotin stock)<br>For BM'''G'''Y: add 100 ml of 10x '''G'''lycerol stock (10%) to final of 1% in the medium.<br>For BM'''M'''Y add 100 ml of 10x '''m'''ethanol stock (5%) to a final of 0.5% 

Filter the media using pathogenic free filter (0.22 um) to further low endotoxin level before culture<br><br>