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Expression L19 using Pichia pastoris

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 :


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

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

3. Expression

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

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

More information about Pixhia pastoris expression system is available on the web at http://www.invitrogen.com.

 

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© 2014 ETH Zurich | Imprint | Disclaimer | 13 December 2005
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