Hello, I have been attempting to express a protein in Pichia pastoris GS115 for some time now. I have just discovered via wesetrn blot that all of my protein is being precipitated/forms into this foam/flock at approx 60-72 hours of culturing. No protein is detected in the supernatant prior to this (T24 or T48), it all appears towards the end of the culturing in this flock/foam...
I have two questions:
1) Is it possible to purify my protein from this foam/flock? It is His tagged and usually I would perform IMAC purification but I am unsure if it will work with this sample type...
2) Is there a way to prevent this from happeneing and instead keep the protein in the supernatant for easier purification?
Any and all insight or advice will be greatly appreciated. And I can provide more information if needed. Thanks a million, Laura.
It seems odd that you do not see expression prior to 60-72hr of induction. What is the source you are analyzing via WB? Straight culture media (without cells)? Or are you using IMAC to purify the protein at the early time points?
How fast/slow are you shaking the culture and are you using a baffled flask? What size flasks and media to air ratio are you using for culturing?
I assume your protein is soluble since you are using the secretory pathway of Pichia?
I apologize for the bombardment of questions.
Hi Tom, thanks so much for the reply.
I am using straight culture media (cell free) via WB, and then I took some of the foam/flock and loaded that onto my gel which is where I saw all the signal. I have seen some expression of my protein in the soluble lysis fraction at T24 and T48, but it's not a strong signal.
I was shaking a 300ml culture in a 2L baffled flask at 250 RPM in an attempt to get a lot of aeration to promote protein expression. Yes the protein is soluble I believe.
Thanks again for your help, let me know if you have any other questions!
Hi Laura,
Thanks for the information.
Have you tried to manipulate any of the induction conditions to determine if you can prevent the protein from precipitating? Such as:
1. Increasing the methanol concentration to 1%?
2. Decrease the shaking speed?
3. Increase the baffle flask size to 4L but keep the induction media volume the same?
4. Lower the phosphate buffer to 50mM?
I haven't encountered a protein precipitating in the media when using Pichia previously so I'm just pitching ideas of what I would potentially do differently.
Hi,
I would recommend to use antifoamer. It should increase protein production in general and it should at least decrease production of foam, so that your protein doesn't stick to it.
Article Beyond de-foaming: The effects of antifoams on bioprocess productivity
Hi both,
Thank you very much for your suggestions, I will apply them all and hopefully sort out this problem!
Thanks again,
Laura
You may try to change your tag from His-tag to MBP or mainly GST-tag. They will promote solubility if the abovementioned suggestions do not work for your case...
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