protein expression in Pichia pastoris - (Jul/01/2004 )
'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.
Gongzuo on Oct 23 2004, 01:56 AM said:
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
i think you can try to incube in different temperature....that can help you in getting an overexpression of protein...
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
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
celvas on Fri Jul 2 11:42:39 2004 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.
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.