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Pichia pastoris is a methylotropic yeast used to express high amounts of protein. Secreted expression is achieved with a cleavalable faktor. To yield? high expression strains it is important to select for multiple copies of the insert in the pichia genome. This is achieved with selection on high Zeocine concetration plates (2g/ml).

Material :

• Glycerol or YPD agar plate containing Pichia pastoris
• YPD Medium (Yeast Peptone Dextrose)???????????????????????????????????????? 600ml
• Zeocine (stock solution 100 mg/ml)??????????????????????????????????????????????? 0.6 ml
• BMMY medium (Buffered Methanol complex medium)???????????????? 600ml
• anti-foam? agent
• autoclaved 2l flasks

Procedure :

Strike out? some glycerol on a YPD+0.1mg/ml Z agar plate. Pick up one colony and start to grow the culture.

1. Growing culture

• Inoculate a clone in a sterile 2l flask containing 600 ml YPD + 0.1 mg/ml Zeocine. Use anti-foam agent if necessary.
• Shake the culture for 2 days at 30°C and 250 rpm

(the color of the culture should be white and the OD should be > 10). 2. Induction

• Centrifuge the culture at 3000g for 5 min.
• Resuspend the pellet in 500 ml BMMY (containing 1% MeOH) in a 2l flask.

3. Expression

• Shake at 30°C 250 rpm for 3-5 days. Use anti-foam agent if necessary.
• Add 1% MeOH to the culture every 24h

(During the expression the color of the culture is white and the OD reaches 60). 4. Purification

• Purify the supernatant following the protocol for? the purification of E.coli derived L19 scFv.

Media recipes:

YPD : 1% yesat extract 2% peptone 2% dextrose 2% Agar if agar plates are needed

Dissolve 10g yeast extract and 20g peptone in 900 ml water (20g agar if needed). Autoclave for 20 min on liquid cycle. Add 100 ml 20% dextrose

(add Zeocine if necessary)

BMMY : 1% yeast extract 2% peptone 100mM potassium phosphate buffer pH 6.0 1.34% YNB (yeast nitrogen base) 4x10-5 biotin 1% methanol

Dissolve 10g yeast extract and 20g peptone in 700ml water. Autoclave 20 min on liquid cycle. Cool to room temperature then add the following and mix well:

100 ml 1M potassium? phophate buffer pH 6 100 ml YNB 10X 2??? ml? 500X biotin 100 ml? 10% MeOH Attention: Zeocine has a shelf life of approximately 2 weeks at 4°C Bacterial Zeocine selection requires low salt concentration

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