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Protein Expression in yeast


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#1 biocrazy

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Posted 13 May 2011 - 05:05 AM

Dear Friends,

I am here to seek your help regarding heterologous protein(from plant source) expression in Yeast. I used pYES-DEST52 vector (gateway cloning) and S.cerevisiae EGY48 competent cell. The protocol as follows

1. Inoculation in SD medium + 2 % Raffinose
2. ON, 200 rpm, 30 oC
3. Subculture into SD medium + 2 % galactose (since vector is gal inducible system).
4. Collected 1 ml of culture from 0, 4, 8, 12, 16,20, 24 h
5. Isolated protein using the yeast protein extraction kit from GE healthcare.
but i could not see the expressed protein, even i could not find any signal even in western blot.

If anyone could help me then it will be very kind of you.

-Selvam Ayarpadikannan

#2 truta

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Posted 26 May 2011 - 12:52 AM

Dear Friends,

I am here to seek your help regarding heterologous protein(from plant source) expression in Yeast. I used pYES-DEST52 vector (gateway cloning) and S.cerevisiae EGY48 competent cell. The protocol as follows

1. Inoculation in SD medium + 2 % Raffinose
2. ON, 200 rpm, 30 oC
3. Subculture into SD medium + 2 % galactose (since vector is gal inducible system).
4. Collected 1 ml of culture from 0, 4, 8, 12, 16,20, 24 h
5. Isolated protein using the yeast protein extraction kit from GE healthcare.
but i could not see the expressed protein, even i could not find any signal even in western blot.

If anyone could help me then it will be very kind of you.

-Selvam Ayarpadikannan


Hi!
Are you sure the plasmid is correct, i.e, right orientation relative to the GAL promoter?
How did you transform it into yeast?
Do you keep the selective pressure by always growing them in -URA media?




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