26 replies to this topic

[celvas]

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

Edited by celvas, 22 February 2005 - 08:17 AM.

[Martorse]

Gongzuo, on Oct 22 2004, 04:56 PM, said:

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.

[canoeli]

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?

[Commander8x]

Hi to all,

I´ve 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 what´s 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; I´ve used PmeI.

I have not tried other vectors than pPICZ and other integration strategies yet, but multicopy integration (G418 or HIS selection, I´m 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!

[urequino]

Martorse, on Dec 15 2004, 01:50 AM, said:

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?

Hey, same problem. I cant see any of the replies for your post. How did you solve it, any advice?

[inducer]

'Gongzuo'
Hi, Are able over come your troubles.. I`m newt into the pichia expression family.. if You are able overcome, can u please explain how did u managed it. Thank you.

[Maria Zd]

Gongzuo, on Oct 23 2004, 01:56 AM, said:

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.  
Sam

Did you or someone ever used YPD plate without zeocin..if yes, what was the outcome?
and is it really cruicial to use bacto-agar instead of some other agar? I just need to transfer some clones n it wouldn't take more than few days, so I was thinking to use YPD plates (w/o zeocin n non-bacto agar)..
recommend n oblige

[lola]

i think you can try to incube in different temperature....that can help you in getting an overexpression of protein...

[serhuy]

Hi, just share with you all my experience,

I used pPICZalphaA, and my recombinant protein is expressed better (higher yield) at 20 and 15, compared to 30 degree C as culture temperature during methanol induction. Plus, I used 1% of methanol for induction.

Have fun with Pichia pastoris                                                     

[sharky04]

Hello,

I am new to using Pichia as an expression system and although I working with someone who has worked with Pichia for some years now, we are still having issues expressing my protein and i was wondering if any of you could help me. I am using the pPic3.5K vector in the SMD1163 strain. Although I know its easier/better to have your protein secreted out of the cell, due to the fact that my protein is aggregation prone, we are trying to express it intracellularlly. I have to use G418 for selection but have had no luck due to the fragile nature of SMD1163 and therefore have used the His gene for selection. Right now i am doing a small scale induction in order to try to find clones expressing my protein. In order to do this, I pick my clones, put them in glass tubes with YEPD and spin them overnight in a tube rotator at 30C. The next day, I wash them with H20, add BMMY in order to induce the cells and put them back into the rotator at 30c for about 24hrs. Then I harvest the cells directly into my sample buffer using glass beads. I have gotten two possible "hits" on my westerns but when i have re-done the western to verify there is no expression anymore.  I have also tried to use the X-33 strain but have gotten similar results. Any suggestions/tips you guys could provide would be great!

Thanks,
Monique

[melodie]

celvas, on 02 July 2004 - 03:42 AM, said:

Although we use pPICZalphaC vector for extracellular expression I think these parameters I have told you can affect your protein expression.

You can see protein degradation with western blot. If there are additional bands to those of interest they should be degradation products that compromise the yield of your r-protein.
If these band exist you must be sure they are not caused by western antibodies inespecificities; check this by loading sample controls with no r-protein expression.

Hi ethusiast,

I am a newbie to protein sciences, also using the same vector as urs to express secreted proteins. can i knw normally how u store the expresssion supernatant? (directly store at -80 deg w/o buffers will cause degradation??)and
wat method u used to concentrate the secreted proteins? ammonium sulphate precipitation??and how do u store it?
my protein size is about 49 kDa, bt i hv no idea on hw to select a dialysis membrane for that, any suggestion???

Thanks so much if u cn gv some advice to me.

[sick of labwork]

Dear all,
I have been facing the following problem for almost half year,so i desperately hope that anyone of you could give me some idea to troubleshoot.
I have cloned my insert into EcoRI and KpnI region of pPIC6alpha A vector, the recombinant plasmid was linearised by PmeI and transformed into pichia pastoris SMD and X-33 strains.
1)Transformation was done by Biorad genepluserII (1.5 kV, 25 uF, and 200 ohm)
2)Transformants were plated on 300ug/ml blasticidin YPDS and incubated 3days at 30 deg, colonies grown were restreaked on 500 ug and 1000ug blasticidin antibiotic YPD.
3)Colonies grown on 1000 ug antibiotic YPD were inoculated into YPD broth with anitbiotic and cultured for 18 hr at 30 deg.
4)Genomic DNA was extracted and used as template for PCR using AOX1 forward and reverse primers.
5)PCR result always showed 2 bands in which one is my gene (1.05kb plus the AOX1 region 500 bp) and another band with larger band size (2kb)
6)Expression was performed at 28 deg, 280 rpm for continuous 3 days. Culture supernatant was concentrated 100X by spin coloumn with MWCO 10kDa.
7)SDS-PAGE and WB were performed under optimized conditions.

**Everything seems OK but why am i COULD NOT get any protein bands on WB and no expression??
Is it because of the integration is juz into one of the Pichia chromosome? what should i do to increase the intergration into both chromosome?
Meanwhile, the Intergrated gene  always disappear after a few days of cultivation, what should ido to make it stable within the genome????


Hope to hear some brilliant ideas from you.
thank you.