protein expression in Pichia pastoris - (Jul/01/2004 )

Hi.
YPDS plates? Im not sure what the "S" stands for, can you tell me? I used YPD plates and my clones grow fine.
With transformation, I used electroporation is Ppic9 and worked well... protein can be seen on dot blot but not on western, damn!
Anyone have any ideas?

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Hi all

I only would want to know about problems which people have with proteolysis of expressed protein in P. pastoris and if someone have achieved avoid it how did it.
I know casaminoacids, EDTA, changing pH, protease deficient strains but none of these methods have avoided proteolysis of my expressed protein.
I am limited to work with baffled flasks.

Thanks in advance

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Hi,
S stands for sorbitol
I'm working with Pichia about two years, but I face a similar expression problems with others. My recombinant protein is expressed inconsistently. Now, I'm trying to express it in different culture conditions also. But there are quite a lot of factors, which are more important in expression?

Second, my recombinant protein is attached to his tag, but I failed to purify it through affinity columns. Any ideas?

Thanks!

Have a nice day!
SerHuy

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Hi,

I am working on Pichia with PICzc vector, because my protein works in Golgi membrane so I decide to express intracellular. I have gone though all construct and now I am working on expression part. Currently, I have a problem with the expression. There is no expression at all based on western with c-myc antibody on both GS115 and KM71H strains. I have tried to adjust the condition such as increase methanol but nothing work.
For my condition I use MGYH for increase cell mass, MMH for inducing.

There is one funny thing which very sad to me. At the first time I did the western with the first induction Pichia, It worked very well with nice weatern band on the film. But no more after the first time. It's gone.

Anyone could give me suggestion for get it to work.
--Any particular condition changing?
--Protein extraction method as my protein stick in the membrane
--How many colony that you normally screen to find a nice perfect clone, I haven't screened so far

For Commander8x

How big is the small band that you mentioned. As I aware of only antibiotic gene has been transformed. For the inserted of your gene, could it be possible to have a various size of band from maltiple insertion in yeast genome?

Thank you very much for your help.

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Hi.

I work with P. Pastoris and i reach to express my protein (a chitochine).
I grow P.Pastoris in YPD for 6 days at 30°C . First two days I give glicerol 20% so i only grow my yeast and I havent expression protein. Then I give it 2% of MetOH absolute.
So I can express about 1mg/l of protein (working perfecly)

Someone can tell me as I can improve my performance?

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I am working with pPICZalphaB and pGAPZalphaB plasmids. I managed to transform my gene into P. pastoris through pGAPZalphaB but the activity of my enzyme was low. I tried to transform into other strain of P. pastoris but was unsuccessful. I managed to get colonies on YPDS+zeocin plates but no band was detected when I run the colony PCR. Any idea why is this happening? This happens for both pPICZalphaB and pGAPZalphaB recombinant plasmids. Please help....

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Hi,
S stands for sorbitol, the Invitrogen manuals says that it decreases the osmotical sensitivity of your cells. Otherwise they can die for Zeocin...
Sandri

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Dear all,

I am trying to express a plant protein in Pichia pastoris X-33 strain.
Two months ago, I did the transformation using pPICZB and I selected for multicopy clones using increasing amounts of zeocin (200, 500 and 1000 ug/ml) in YPD plates. Then I checked them by colony PCR using the primers 5AOX1 and 3AOX1 and I observed three bands: one of 3,0 kb corresponding to the size of my insert (2,7 kb) plus the 300 bp of the vector, a 2,2 kb corresponding to the AOX gene in the other locus and another band near 300 bp which I don´t understand.
I ckecked for protein production in a small scale using 5 ml culture by western blot with an anti His tag Ab and I get expression of the protein. So I save this clones as glycerol stocks.
I have just now start again this work and now I can´t get the protein expressed and when I checked by PCR I can´t see the upper band which I saw before.

My question is: Is it possible that I didn´t obtain stable transformants before? Could have happened that the plasmid was in the genome and that is why I did have a correct band with colony PCR? I don´t undestrand. Does somebody have a quick method for isolation of genomic DNA of yeast so I do that instead of the "quicker" colony PCR?

Thanks very much for your help!
With hopes of your answer soon!

llsb

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Hello,

I'm using Pichia pastoris (Invitrogen) to express glycosyl hydrolase enzymes
from Arabidopsis. Has anyone expressed something similar? I'm having
problems expessing my protein. I'm using pICZA vector, 25 ml cultures in
baffled flasks, complex glycerol/methanol media, 4-5 day induction w/ 1%
methanol. Transformants were determined by colony PCR. Various other native Pichia proteins are being expressed, and Western blotting (using Sigma H1029 anti poly-His antibody) does not pick up
any band of appropriate size in pellet or secreted fractions (secreted
fractions are TCA precipitated). The antibody works well for a bacterially expressed
recombinant C-term His tag protein. Control human serum albumin secreted
beautifully, so I can not say my technique is incorrect. Does anyone have any
suggestions. I'm willing to give more detailed information if requested.
Thank you!

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Hello,

How many clones have you tried?

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