I´m trying to express one product in Pichia pastoris but this organism is cleaving it. I added PMSF to the expression medium but it was not effective. Did anyone have this problem?
Causes of Proteolytic Degradation of Secreted
Recombinant Proteins Produced in
Methylotrophic Yeast Pichia pastoris: Case Study
With Recombinant Ovine Interferon-T
http://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1001&context=chemengbiochemeng
PMSF is acting as antiprotease but is not sufficient you should add other molécules such as benzamidine also EDTA but i recommend you to try à cocktail containing antiprotease (sigma sells one, but you can find other companies). You will target many proteases this way.
I would recommend to add not just one protease inhibitor like PMSF, because it inhibits only serine proteases, but add protease inhibitor cocktail containing the inhibitors of all different proteases (acid proteases. cysteine proteases, metalloproteases, serine proteases, and so on). Another suggestion would be not to add anything to your expression medium, it's not going to help. You need to add your fresh solution (without any previous thawing/freezing or incubation in solution for some time) of protein inhibitor cocktail to your cell lysis buffer immediately before the cell lysis step. When you make cell lysis, a lot of different proteases are released from their natural sub cellular contained locations. That's the moment when your expressed protein is mostly vulnerable to proteolysis. Here is the info from two different commercial sources where you can get the protease inhibitor cocktail. You just need to decide if you need EDTA in these cocktails or not. The presence of EDTA inhibits some metalloproteases, but if your protein needs some metals for it's function, the EDTA might inactivate your protein.
http://www.sigmaaldrich.com/catalog/product/roche/11836170001?lang=en®ion=US&gclid=CJ7S5bGNl84CFQmKaQod-RMO5A
https://www.thermofisher.com/order/catalog/product/78430
Are you attempting secreted or cytoplasmic expression of your protein? If your problem is degradation, then adding an inhibitor cocktail at the time of cell breakage (for cytoplasmic expression) may be effective. If, as I presume from your post, you're trying secreted expression, then using such a cocktail may turn out very expensive in the larger volume of the culture medium (and possibly ineffective). Furthermore, addition of a serine protease inhibitor (such as PMSF) may inhibit the proteolytic release of your recombinant protein from the signal peptide, thus preventing its secreted expression.
What evidence do you have that cleavage is occurring? Could your problem be truncation of translation? Is your gene codon-optimised for Pichia?
What is your expression temperature? I've read that lowering the expression temperature (to 20 degC, for example) can improve expression levels. This could conceivably be related either to facilitating correct folding, or reducing proteolytic activity.
If the problem is indeed degradation, a colleague here with more experience of Pichia expression suggests altering the pH of the growth medium (to reduce protease activity), or the inclusion of a rich protein source (such as casamino acids) to give the protease(s) an alternative substrate to keep them busy.
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