Mysterious protein degradation - (Jul/11/2008 )
Hi all,
I am quite desperate, and so I am turning to this forum in an effort to get some new ideas. I have a serine recombinase that has a C-terminal kinase tag (for radio-labeling with gamma 32-P) and a hexa-histidine tag (for purification purposes). While I can purify the protein beautifully and label the protein with the kinase just fine, the protein begins to degrade once I add it to the reaction!!!
Basically, the protein itself, once labeled and run on SDS-PAGE is very clean (just my band of interest and one other small product that I cannot identify) - but once I add the buffer (consisting of glycerol, Tris, EDTA, and O-glutathione), the protein degrades in a matter of hours.
I have tried to add protease inhibitors, but as the protein is a serine recombinase, a serine protease inhibitor inhibits the protein!
I just tried cysteine protease inhibitors (E-64) and pepstatin A, as well as PMSF (hoping that the short half-life might help prevent degradation but not inhibit the reaction), but no luck. I have tried adding BSA as well (to stabilize the protein), but no luck.
The only luck I have had is if I use a protease inhibitor cocktail from Sigma that contains AEBSF etc, but this cocktail inhibits the reaction!!!!
I'm really confused, as the protein itself, when labeled and incubated (on its own) at 37C does not degrade, but adding reaction buffer causes it to degrade quite rapidly! This would indicate that the protein itself does not contain a protease, but the reaction buffer does. I have autoclaved and filter sterilized all components of the reaction buffer, with no luck.
Any suggestions would be VERY much welcome!!!!!
--miss phd
Sounds like you might have something in Nature or Science magazine. An inducible self-cleaving protease? Hope it is not a naturally occurring protein but you created. This thing will be extremely useful if an enzyme can be engineered to have this self-limiting property - just add a mild stop buffer to induce its cleavage the reaction is stopped; or if a protein drug's activity (either its therapeutic or toxic effects) can be stopped by taking a 2nd pill! CONFIRM your finding first, and then dig it up and it could be a big project for you.
I am quite desperate, and so I am turning to this forum in an effort to get some new ideas. I have a serine recombinase that has a C-terminal kinase tag (for radio-labeling with gamma 32-P) and a hexa-histidine tag (for purification purposes). While I can purify the protein beautifully and label the protein with the kinase just fine, the protein begins to degrade once I add it to the reaction!!!
Basically, the protein itself, once labeled and run on SDS-PAGE is very clean (just my band of interest and one other small product that I cannot identify) - but once I add the buffer (consisting of glycerol, Tris, EDTA, and O-glutathione), the protein degrades in a matter of hours.
I have tried to add protease inhibitors, but as the protein is a serine recombinase, a serine protease inhibitor inhibits the protein!
I just tried cysteine protease inhibitors (E-64) and pepstatin A, as well as PMSF (hoping that the short half-life might help prevent degradation but not inhibit the reaction), but no luck. I have tried adding BSA as well (to stabilize the protein), but no luck.
The only luck I have had is if I use a protease inhibitor cocktail from Sigma that contains AEBSF etc, but this cocktail inhibits the reaction!!!!
I'm really confused, as the protein itself, when labeled and incubated (on its own) at 37C does not degrade, but adding reaction buffer causes it to degrade quite rapidly! This would indicate that the protein itself does not contain a protease, but the reaction buffer does. I have autoclaved and filter sterilized all components of the reaction buffer, with no luck.
Any suggestions would be VERY much welcome!!!!!
--miss phd
Have you tried other buffers?
It could be a big issue, haha. But right now he need to stablize the protein for assays
I am quite desperate, and so I am turning to this forum in an effort to get some new ideas. I have a serine recombinase that has a C-terminal kinase tag (for radio-labeling with gamma 32-P) and a hexa-histidine tag (for purification purposes). While I can purify the protein beautifully and label the protein with the kinase just fine, the protein begins to degrade once I add it to the reaction!!!
Basically, the protein itself, once labeled and run on SDS-PAGE is very clean (just my band of interest and one other small product that I cannot identify) - but once I add the buffer (consisting of glycerol, Tris, EDTA, and O-glutathione), the protein degrades in a matter of hours.
I have tried to add protease inhibitors, but as the protein is a serine recombinase, a serine protease inhibitor inhibits the protein!
I just tried cysteine protease inhibitors (E-64) and pepstatin A, as well as PMSF (hoping that the short half-life might help prevent degradation but not inhibit the reaction), but no luck. I have tried adding BSA as well (to stabilize the protein), but no luck.
The only luck I have had is if I use a protease inhibitor cocktail from Sigma that contains AEBSF etc, but this cocktail inhibits the reaction!!!!
I'm really confused, as the protein itself, when labeled and incubated (on its own) at 37C does not degrade, but adding reaction buffer causes it to degrade quite rapidly! This would indicate that the protein itself does not contain a protease, but the reaction buffer does. I have autoclaved and filter sterilized all components of the reaction buffer, with no luck.
Any suggestions would be VERY much welcome!!!!!
--miss phd
Have you tried other buffers?
Dear Durandal
Do all your purifications at cold conditions. it seems there is a problem in reaction buffer did you try any other buffer ? and Labeling also matters , how you are lableing the protein is it correct confirmation lableing , and how u are proving that your enzyme is going in degradation (because of less activity or any gel you have run or by what basis).
Correct me if iam wrong
Regards
S