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

I cannot tell you right now, I taught it was written in the expression protocol. For growing them up just before expression, you need buffered minimal glycerol medium (BMG). For expression you need medium with methanol (if you go for the AOX promoter), I used buffered minimal methanol medium. For both the pH was 6.0.
You can also use the unbuffered ones, the medium then becomes more acidic, this can inhibit proteases a bit. If you want to express it intracellular, I would go for buffered medium.

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Thanks . I found out that the minimal medium is better for secreted proteins as there is less ingredients and purification gets much easier. My pichias express intracellular. Anyway I´ll try the minimum media and see if it works better than buffered full medium.

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I've only joined this forum, so 4give me for referring to things past. Regarding your message below:

can you pls send me your SDS gel photos & western blots! It seems I'm getting unspecific binding when using a histidine probe as well as a monoclonal Ab.

thanx

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Hello everybody!

I'm looking for a reliable protocol to do direct PCR screening of integrants into the pichia genome.

First I tried the one provided by invitrogen that did not work. then I tried several things and could get finally a result with normal colony PCR. However, I could never repeat any usable results. Sometimes I get a band around 250-300 bp, but I get this also with pichia transformed with empty pPICZA.

any hints or suggestions?

Thanks in advance!

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Have you designed primers with one primer just outside the place where it should integrate and the other primer inside your construct (inside your insert in the pPICZA)?

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I only used the 3' AOX1 and 5' AOX1 primers...

The primers you describe, do they work better than the AOX primers and why?

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How big is your insert? It might be to big for PCR with the AOX primers. I've never worked with the AOX primers.
Since you sometimes get the same fragment with the empty vector, I would go for multiple approaches.
If you take the AOX primers, you should get a fragment higher than the empty vector. But if you take 1 AOX primer and one in your insert in the vector, you will only get a fragment if your insert is in the vector (or still in the vector. I assume that you have checked if the insert was in before transformation, but there is always a small chance of recombination). Do you understand what I mean? I'm not sure if I'm clear. So with the other primers I mean a primerpair with 1 AOX primer and 1 you have designed yourself on a piece inside your insert.

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hi all,
my gene is in pPIC9K , protein comes in bmmy media. I used to get good expression until few months back, but not anymore!

Albumin control is giving good amount of protein in the in the media (which means media is allright).
my pichia pastoris clones are growing in MD media (which means hisgene is present).
But still i have no protein in BMMY media.

my clone grows verywell in BMGY. Expression is under 1% methanol ,300 rpm, 28 degrees (this EXACT conditon used to express my protein in good amount)
is there any mutation that prevents the secretion or expression itself?
waiting for help!
jughead.

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I did PCR also with primers that anneal inside my gene, but did not work. I also tried the protocol from "pichia protocols", but used lyticase instead of zymolyase... Well, I think I will just abandon this PCR screening.


Today I found that my protein got expressed in high amount, but insoluble (clone that grew on 500 ul/ml Zeocin, in MM, 30°C, 250 rpm).
Does anybody know what one can try to get soluble expression? Maybe find a MutS-clone? or lower the T? or what else? does another medium such as BMM change something?

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did you use old colonies from the plate? or how did you restart your expression?

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