Protease Inhibitors? - I want to know what are the best protease inhibitors to yeast. (Jul/10/2008 )
Hello, I am using Pichia pastoris to overexpress a mannosiltransferase from C. albicans. I used the strain X-33 and the vector pPICZalphaC. I got the protein but after 48 h I see 3 bands in the gel indicating protein degradation. I tried the medium BMMH peptone, yeast extract, biotin, methanol, YNB w/o aminoacids and potassium buffer pH 6.0 plus protease inhibitors (coktail tablets from Roche) but the proteases activity was not complete inhibited. I tried to use 1% casaminoacids in the medium and also I used an unbuffered medium but did not work.
NOTE: the protein is secreted to the culture medium
I am kind of desperate. I hope you can tell me what is the best protease inhibitor to yeast proteases.
Thank you in advance
as you undrestand proteins are not stable in medium they usually degredated up to 90% in the first 15 minute so check your cocktail to be correct and not expired.
cheers,
akhshik
two quick suggestions:
1: Roche protease inhibitors can be used at twice the concentration if needed...although you might be doing that already...
2: There's a protease inhibitor from Sigma that I use that seems to work well... I can't remember the part number but just do a search on protease inhibitor coctain in the Sigma-Aldrich webpage..
it contains the following:
aminoethyl-benzene sulfonyl fluoride
aprotinin
bestatin
E-64
leupeptin
pepstatin
calpain inhibitor I
When in your buffer you want them each at a concentration of:
0.2 mM aminoethyl-benzene sulfonyl fluoride
1 microgram/ml aprotinin
10 microM bestatin
3 microM E-64
10 microgram/ml leupeptin
2 microM pepstatin
50 microgram/ml calpain inhibitor I
Also, you don't have to buy a cocktail. You can simply make them yourself (I say simply but...you know what I mean). it's possible that what you make will work better.
3: (okay THREE suggestions)
Always, Always, Always, keep the cells and buffer and lysate COLD. work in a cold room if you have to.
I hope this helps
1: Roche protease inhibitors can be used at twice the concentration if needed...although you might be doing that already...
2: There's a protease inhibitor from Sigma that I use that seems to work well... I can't remember the part number but just do a search on protease inhibitor coctain in the Sigma-Aldrich webpage..
it contains the following:
aminoethyl-benzene sulfonyl fluoride
aprotinin
bestatin
E-64
leupeptin
pepstatin
calpain inhibitor I
When in your buffer you want them each at a concentration of:
0.2 mM aminoethyl-benzene sulfonyl fluoride
1 microgram/ml aprotinin
10 microM bestatin
3 microM E-64
10 microgram/ml leupeptin
2 microM pepstatin
50 microgram/ml calpain inhibitor I
Also, you don't have to buy a cocktail. You can simply make them yourself (I say simply but...you know what I mean). it's possible that what you make will work better.
3: (okay THREE suggestions)
Always, Always, Always, keep the cells and buffer and lysate COLD. work in a cold room if you have to.
I hope this helps
Thank you, the problem is that I need to add the protease inhibitors since I place the cultures to grow because the protein is secreted by the cells to the culture medium and is a big volume 250 mL. I am really considering to make my own cocktail because is cheaper I tried the Roche cocktail but does not content inhibitos to aspartic proteases.