Does my protein degrade during DNAse I and thrombin digestion? - (Apr/16/2007 )
I have two problems when purifying GST-fused protein and I used the B-PER GST fusion protein purification kit from Pierce to do that.
Firstly, after I add B-PER reagent to lyse E.coli at room temperature for 10 min, sometimes the solution appears to be viscous. The manual tells me that it should be due to genomic DNA interruption and recommends me to use DNAse I to digest DNA. But the problem is that DNAse I works at 37 degree for 30 to 60 min, if that is the case, will my protein degrade at 37 degree?? Or can I do that at room temperature, how long?
Secondly, once I get purified my target GST-fused protein, I need to remove the glutathione part by thrombin digestion since my vector is pGEX2T/4T and only thrombin works. I check the Amersham website, that enzyme works at 22 degree for 16 h, so if I do that, will my protein degrade at that temperature.
I am really new to protein purification, so I really neeed your guys' help, thx!!
-Jason
Firstly, after I add B-PER reagent to lyse E.coli at room temperature for 10 min, sometimes the solution appears to be viscous. The manual tells me that it should be due to genomic DNA interruption and recommends me to use DNAse I to digest DNA. But the problem is that DNAse I works at 37 degree for 30 to 60 min, if that is the case, will my protein degrade at 37 degree?? Or can I do that at room temperature, how long?
Secondly, once I get purified my target GST-fused protein, I need to remove the glutathione part by thrombin digestion since my vector is pGEX2T/4T and only thrombin works. I check the Amersham website, that enzyme works at 22 degree for 16 h, so if I do that, will my protein degrade at that temperature.
I am really new to protein purification, so I really neeed your guys' help, thx!!
-Jason
Hi jason.
Sorry to be of little use to you, but you're just going to have to try it and see. I have digested a fusion protein and had no problems, then tried a related protein and had massive digestion.
May I suggest you try a time-course expt? Set up a digest, and collect samples to run on a gel, say every 15 minutes. Try and get some of the beads as well, spin them down, separate beads from S/N, stop the reaction with SDS loading buffer and boil. You should be able to run both S/N and beads, so you can see the fusion, as well as GST and your protein.
All the best.