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Dear all,
I have tried to express my protein - sucrose synthase from rice, in [/I]P.pastoris. Although I tried both electroporation and " Easy Comp" from Invitrogen, I could not get over expression of my protein.
Here are my inducing conditions:
30 C
150 rpm
medium BMGY FOR 16~18hr
change to BMMY OD600=1
used 0.5% methanol for induction
I am curious that should I try higher methanol for induction or try to incubate in different temperature?
Or how can I get an over expression of my protein?

thanks

Hi,

you say you could not get overexpression but did you get any expression?

I am working too with Pichia p. and there are several factors which can affect the expression of your protein.

Hi again,

as you say you have poor expression of your protein since you have low level signal with western blot.

About the growing parameters I think 300 rpm is better than 150 rpm.

Also, some proteins are better expressed with higher amounts of metanol, try 1% methanol instead 0.5%.

Another interesting parameter is degradation of the recomb-protein. Did you see some amount of proteolysis in westblot?

Other possibilitys are related with plasmid. What kind of vector did you use? Is your protein secreted or not? Was your plasmid completely linearized before yeast electroporation?

About your purification problem I'm not sure if I have understood you. You say your encoding gene has a stop codon before HisTag. If so, you can not hope your protein has been produced with HisTag because protein codification finalizes with a stop codon. Therefore you can not use chromatographic techniques related with HisTag proteins.