Missing expression of proteins of interest in Pichia pastoris - (Jan/30/2012 )

Hi all

Hope that some of you might be able to help me out - I am seriously frustrated!
I am working on expression of two different plant lipid transport proteins (LTPs) in Pichia pastoris. They have a size just below 10 kDa. I have cloned the sequences into the pPICz(alpha) A vector from invitrogen (secreted expression) and confirmed the success of this step with sequencing of the constructs. Then I have transformed the linearized vectors into P. pastoris X33, and confirmed that the resulting transformants posses resistance to Zeocin. Furthermore, I have used different PCR screenings to confirm the correct integration of the constructs into the pichia genome.
So far so good.
Then I have chosen one transformant for each construct which has shown positive PCRs. I have performed expressions according to the Invitrogen protocol: First I grew up the cells in BMGY media, then shifted to BMMY media with induction with methanol every 24 hr and 250 rpm. I have tried growing them for 96 hours and 72 hours, and I have tried varying the temperature (23-29 degrees). The cultures seem to be growing just fine at all these conditions, and I have previously expressed (bigger) proteins in Pichia, using the same condition and system, without problems…
Furthermore, I have no antibody available to do a Western blot.

So now for the current problems:
1) I seem to have a very low general amount of protein, both in the background culture and the transformants. In lyophilized supernatant from background and transformant cultures I only have approximately 0.2% total protein present in the powder. Furthermore, I almost can’t see anything on SDS PAGEs, even when using silver stain. Bands are only visible at the top of the SDS PAGE, which then gets more and more unclear, and ends up in a smear.
2) I cannot confirm the presence of the proteins of interest on an SDS PAGE –possibly because they simply are not there. I have been using 10-15% glycine SDS PAGEs. I am soon to try a tricine SDS PAGE, but I believe I should be able to see something on the glycine SDS PAGE?!
3) IF the proteins are not expressed – how can this be? Is it possible that expression just does not occur, even though everything seems to be correct? Any suggestions what to do now?

I am in a bit of a pickle… I have a very limited time schedule to get success with this expression; else I have to move on to another project. Therefore I hope that someone can give me some good advises or hints 

Thanks in advance!
- Katrine

Hi Guys,

Could you please let me know the reason for using some expression cassettes in the forward orientation and some in the reverse in Plant expression constructs?

Thanking you.

Many reasons could cause low or no expression.

Impropal transgene sequence design could cause expression low, for example, the bad design may cuase transcription difficulty due to un-prefered codons, mRNA secondary structure, or mRNA unstable, etc. During transfromation, the integration may happen accidently at a undesired locus which may cause gene silience, or the multiple integration may also cause gene silence. Another possibility is the protein you are expression is very sensitive to some endogeneous protease, or just simply not stable, and they degraded before you collect them. Or it is just the protein itself, some protein just expresses better than others without clue of why.

What I would suggests are follows. Grow at least 10 transformants for each strain to evaluate their expression because each transformant likely have different integration events (locus, integration times, etc.). If problems still persist, then try some Pp strains with protease deficiency (not sure whether commercially available). Finally I would suggest to optimize the codons of your proteins specifically for Pechia pastoris.

Hope it helps. Good luck!