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

Hi all
Recently, I manage to express one of the serine proteases in P.pastoris . My protein is attached to c-myc epitope and His-Tag. I also manage to purify this protein but here is the big problem. My protein has got very low activity comparing to native one. Has anyone got similar problem? I am wondering is it possible that this is due to His-tag presence? Any ideas what should I do to solve this?


Thank you very much for your help.
kasia

Hi,

I got the similar problem. After the purification step(I used the Ni-NTA spin column - His tag column), I run the SDS-PAGE and stained the gel with coomassie blue stain. I found there are many many bands but I could not find my target. When i did the western blot, I could get a band which size was similar to my target. However, I also got three more bands which size was larger than my target. I used c-myc antibody. I still think how i can improve it. Did u do the western blot?????

siuchi

Yes I did western blot and I run SDS-PAGE. I got only one band which size is similar to native protein. I didn't mentioned before, that I purified my protein using gel filtration. The only problem I've got is with this protein activity, which is very very low.
I have only one idea- to test another conctruct which is without His-Tag and c-myc. Have anyone got better idea than my?

I am using Pichia pastoris to secrete an ezyme extracellularly, I have cloned the gene but i get very little amount of protein. I think my protein is too huge (60 kDa) to be secreted into fermentation medium.Is there comments.please helppppp.

have you checked for positive clones?

The pichia's were able to secrete my fusion-protein of 130 kDa. What are you precizely using?
Because multicopy-transformants were not able to secrete it, I found it intracellularly, but low copy transformants could do it.
Have you already looked in the cell?

Hi Aspergillie,

What vector and yeast strain did you use?

Thx very much!!!!

siuchi

Dear all,

As a thesis subject to get my Bachelor's degree, I'm trying to express a protein in Pichia pastoris. But like many of you, I'm getting some difficulties....

I'm using the pPIC9 vector for secreted expression in the GS115 Pichia strain. The vector was linearized with SacI, and another sample with SalI. As a transformation method I used electroporation. Here I'm wondering what exactly the DTT (dithiothreïtol) does with the cell wall, as it is said to increase electroporation efficiency 150x.

I transfered the cell suspension on MD-plates (selective medium, lacking his) and colonies appeared after 3 days. Initially, I used colony-PCR with the AOX1 sequencing primers to screen for good transformants. Here I only was 1 band: on most colonies a 1,1kb band, corresponding to my gene of interest, and on a few others the 2,2kb band, corresponding with the AOX gene, but never both.

When did PCR on isolated genomic DNA, I got no bands at all. (Not with AOX sequencing primers en not with gene of intrest specific primers) The controle samples (the vector with and without insert) did result in the appropriate bands. I checked the genomic DNA on 0,8% agarosegel and all seemed ok. I already tried 2 diffrent DNA isolation protocols, one with PEG, and one with triton X-100+SDS.

Currently I'm growing a few colonies on BMMY to look for expression anyhow.


Does anyone have an ideas?

Regards,
F.

siuchi,

sorry for my late reaction. I've used the pPICZaA vector. By streaking colonies on plates with increased zeocin concentration, I selected the high-copy and the low-copy transformants. May be due to the size, the low-copy transformants worked better in this case. If I had time, I would have liked to also try a MutS strain, maybe because they grow slower, they could handle to protein even better?

Hi all,

obviously I am not the only one having problems overexpressing my protein in pichia :-)
I have a question about the media. The invitrogen manual tells about 3 different media options (BMGY, BMG and MGY). But they don´t explain witch media is best for intracellular proteins and witch pH is best to start with.

Does anyone know?