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

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QUOTE (dreamcatcher @ Jul 1 2004, 02:59 AM)
Dear all,
                  I have tried to express my protein - sucrose synthase from rice, in [/I]P.pastoris. Althuogh I tried both electroporation and " EasyComp" from Invitrogen, I could not get overexpression 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 incube in different temperature?
Or how can I get an overexpression of my protein?

thanks rolleyes.gif

Apparently protein expression in Pichia is quite sensitive to temperature, and stops above 32C. Invitrogen protocols strongly recommend to grow strains at 28C, to prevent overwarming in cultured flasks (be aware that even if temperature inside the shaker is 30C, the flask content can get warmer due to yeast growth). Wish you luck,
Marta

-Martorse-

I am trying to express a protein in Pichia pastoris. Problem is, I have noticed that growth of transformants in MGY (minimal medium) is really slow or even non-existing when I inoculate them after growth in YPDS. What can I do?

-Martorse-

HI,
anybody can tell me how much linearized DNA u use for electroporation transformation in Pichia.
and How much of circular if any.
thanx

-clone-

Hello

It might be late for an answer but you can download the manual from the invitrogen web site it will tell you exactly how much linear DNA or circular DNA. Just follow what they say that's what I did to teach myself how to use pichia which doesn't I don't have problems but overall it works OK eventhough I wish I could improve my expression yields.

-tweete07-

Hi all,
i'm trying to express my flavoprotein in Pichia pastoris via pPicZalphaA cvector according to the invitrogen protocol but... where is my protein? i cannot see any band corresponding to it in SDS page and western blot!
growth conditions are the suggested ones ... no idea really... any brilliant advice for me?
many thanks by now and hope to read from you.

-gretafaccio-

Hi,

I recommend you to check step by step your proceeding way:

1. Identification of well cloned gene into vector

2. Are your colonies zeozin-resistant?

3. If so, may be the protein is being poorly expressed. Try to concentrate your proteins samples 10-50 fold and try to detect it by western

4. Recheck your culture conditions (time, rpm, media, pH, methanol concentration...)

Good luck

-celvas-

QUOTE (Gongzuo @ Oct 22 2004, 04:56 PM)
Hi, you all,
I am a new worker in this area.  I am trying to express a protein by using Pichia pastoris.  We got the expression kit from Invitrogen.  I already have my target gene cloned in pGAPZalphaB and also in pPICZalphaB.
Now my problem is that we cannot get yeast colonies on YPDS-zeocin plates after electroporation of the competent yeasts with the linerized gene construct.  I have discussed this problem with seveal professional people, but nobody can find where my problem is.  Nobody knows why.
Did anybody get this problem before?
How to make sure that our gene construct can be transfected into the yeast ?
Your answers will be appreciated.

Sam

Gonzuo:
I had some trouble transforming Pichia, and finally managed some good transformants by increasing amount of linealized DNA used in electroporation, Pichia is tricky to transform, you should be using 1-5 ug DNA aprox. Also, minimal amount of resuspending buffer was used (increases concentration). Electroporation is far more efficient than EasyComp kit.
You can check transformants by PCR, using 5AOX and 3AOX primers, purchased from Invitrogen. Good ones would usually give 2 bands: one corresponding to AOX gene and one corresponding to heterologous gene.

-Martorse-

I have bought the multi-copy pichia expression kit. But my transformation of pichia failed for many times. Every time I discovered that GS115 strain grow together to form a lot of small particles wihch contain 10-30 cells because of its budding. Did the pseudohyphae influence the efficiency of transformation? How can I prevent this phenomenon?

-canoeli-

Hi to all,

Ive now read this thread because I also looked for advice to express a protein in Pichia. Thanks to those who told me something new.

Now I have some comments to this theme:

1. Read the literature and be sure to understand whats written there. The Invitrogen manuals are the first and most important source of information. Very well but expensive is also Methods in Molecular Biology vol. 103 ("Pichia Protocols"), edited by D.R.Higgins and J.M.Clegg. The latter is somewhat of a Pichia Godfather, so look for other papers.

2. It is absolutely mandatory to verify the correct integration of your (linarized) gene into the Pichia genome! When U linearize in the AOX1-promoter site, the gene is recombinated into the Pichia AOX promoter site. Hence there is only one on every allele, maximum will be a "double transformant". Do a PCR with genomic DNA from your transformants, and when you see only a long band (depends on your gene), then you have a good clone: a "homozygous double transformant". This is what you want!
Two bands mean integration just on only one chromosome, and a small band means resistance to zeocin, but no integration. These clones will show little or no expression, friends!

3. I use pPICZB and pPICZalphaB to integrate into the X-33 wildtype strain. I did transformation by the LiCl-PEG-heat shock-method. That gives no maximum efficiency, but one cannot handle more than 20-40 clones at a time, right? The advantage is that you use the competent cells immediately after preparing them. Be also sure to choose the right enzyme to linearize; Ive used PmeI.

I have not tried other vectors than pPICZ and other integration strategies yet, but multicopy integration (G418 or HIS selection, Im not sure) is obviously much better.

4. Secretion (by using the pPICZalpha vectors) is favorable because of much easier purification. BUT: the protein can also become oxidized because of contact to air during shaking/fermentation. Any affinity tag is helpful to isolate your protein. If necessary, cleave it with a protease during purification.

5. Fermentation is favorable because of better growth control. In my eyes, the most important parameters are continuous methanol feeding (instead of addition every 12/24 hrs), excessive aeration and better temperature control. This is what my experience shows.

Good luck with expression!

-Commander8x-

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