In the case of Mut+ recombinants, we first grow cells in a small amount of BMGY till the OD reaches 2-6 and then dilute it in BMMY of OD to 1. But in case of Muts we do the opposite, first we grow the cells in large amount of BMGY till the OD reaches 2-6 and then concentrate it to 10 times in BMMY.
Hi,
I don't have experience wth MutS strains, I have always used WT X-33 (or rather, I didn't investigate the phenotype).
To understand, we have to understand the phenotypes (I myself understood them only recently:).
So Mut+ is the WT, which utilizes greatly methanol. It is able to metabolize it and grow solely on methanol (or other C1 compounds).
MutS (for methanol utilization slow) lacks the main alcohol oxidase AOX1. It still has functional AOX2, but it is slower and less efficient.
Then there is Mut-, which lacks both AOX genes and is not able to grow on methanol (or other C1 compound) as sole carbon source.
Thus, if you have Mut+, you grow overnight culture on glycerol, which grows OK and without expression of your POI. Then, you dilute the overnight culture (similarly, like when you grow bacteria) and transfer it into induction medium. Because Mut+ can grow nicely on methanol, you can dilute the yeast and it will grow to high cell densities.
On the other hand, with MutS, which doesn't grow much on methanol, if you'd dilute it, you'd get very low cell density and thus your protein would be at very low concentration. Thus, instead of diluting, you concentrate the overnight culture to reach the high cell density, which is one of the advantages of Pichia.
Tomáš Hluska thank you so much, its really helpful.
I have confirmed that my gene has been incorporated into the WT-X-33, by PCR using 5'AOX1F and 3'AOX1R primers. It gives 2 bands one corresponding to my gene+aplha factor and one corresponding to Aox1 gene, but when I go for expression I am not able to see any expression. I have tried using Casaminoacid and also tried the expression in Unbuffered media. Can you suggest me something I could do.
Sorry for the delay.
How do you detect your protein? Activity, Western blot (against POI, tag), SDS-PAGE, something else?
So what is your protocol for expression?
Is your protein excreted? You wrote about pPICZalpha plasmid in another question, so probably yes. How about trying intracellular expression? How about constitutive expression (e.g. pGAPZ(alpha) plasmid)?
Tomáš Hluska Thank you so much for replying.
Yes my protein should come in the extracellular medium, as I am using alpha factor.
I check for my protein expression by SDS-PAGE, but I did not see any expression using BMMY in secretory form as well as in the intracellular (as I have checked for expression in the cell also) in comparison to uninduced one.
So my first I grow the WT-X-33 containing my insert in YPD, then next day I grow it in BMGY, for 24 hours, then I change the media to BMMY(In this case I have tried both reducing the medium like in case of Muts and as well as increasing the medium as in case of Mut+). I induce every 24 hours with 0.5, 1, 1.5, 2, 2.5% MeoH.
But Just now I tried expressing it in unbuffered medium (YNB+MeoH), I was able to see distinct band around 116kDa but my protein is around 65kDa.
Jyoti Rustagi unfortunately, SDS-PAGE is neither specific, nor very sensitive. What is your protein (could you use any sort of activity measurement?), do you have any tags (Western blot)?
Is there any chance your protein could form dimer? The sizes on SDS-PAGE are only approximate, so apparent 116 kDa could be 65 kDa-protein dimer.
How about trying to delete the alpha-factor? Maybe your protein doesn't like it outside. You could do that easily by this method
Article In vivo cloning of up to 16 kb plasmids in E. coli is as simple as PCR
I can help you design primers and arrange the experiment, if you wish. I'm having some troubles with cloning, but working with single fragment (deleting little piece) works fine.
I assume you induce for 3 days? Did you measure OD600? From that you should know, whether your yeast grows on methanol or not, so you'll know next time, whether you should dilute or concentrate.
I don't see any problem with your protocol. Maybe try to contact Invitrogen, or whatever's their name at the moment.
Tomáš Hluska I'm incredibly sorry for responding so late.
My protein is antibacterial, I can check the activity but its amount is so low.
In SDS do we get the protein in dimer form?????? I am assuming that SDS protein should be in primary structure condition.
Okay, I can try it without the alpha factor as well.
This time I tried purification even after I was not able to see the expression in SDS, then after purification using nickel NTA, (I diluted the 20ml media in 80ml binding buffer and then eluted in 2ml after its TCA precipitation I ran it on SDS) I got 2 bands near 66kDa band of the marker, earlier it was not visible. Then I did in-gel digestion and gave the sample for HR-MS, I am getting the hits related to my protein.
I induce for 4 days (Including the first day in which we change the media). I did not check the OD every day of induction but visually I don't find any significant increase in the cell mass as compared to the day of 1st induction
I tried contacting Invitrogen but they are not responding.
Jyoti Rustagi what is your progress so far?
Yes, protein can remain in oligomeric state even during SDS-PAGE. It's rare, but if the band appears upon induction (it is not present in uninduced medium), you could check it if you have access to MS.
What are your growth conditions? I assume in Erlenmayer flasks? What is the rpm? Pichia is very demanding for oxygen, so shaking at maximum speed is important. If I remember correctly, it is also crucial that the temperature does not exceed 30°C (but I may be wrong about this one).
The advantage of Pichia is that it is able to grow to very high cell densities and hence you get high protein yields. So first, I would focus on the Pichia growth and then you will see, how is the expression. You could try growing on methanol and on glucose and measure the OD600 to see if your yeast is viable.
Tomáš Hluska When i repeated the experiments on same conditions i did not got that prominent band. Yes I am using erlenmeyer flask and i have tried growing cells on 250-300rpm. So right now i am going to start the expression in pichiapink strain 4 in case my protein is getting degraded.
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