Thanks to everyone who responded to my post (especially, John Lee and Brad Bennett) with helpful comments and suggestions. The good news is that I was able to get the Ulp1 protease cleavage step to 100% completion at 4C by using between 1:3-to-1:2 enzyme:substrate ratios and incubating the reactions overnight on an orbital shaker to ensure constant mixing (instead of dialysis).
It turns out that I didn't have to switch from beta-mercaptoethanol to TCEP. I also kept NaCl concentration at 150mM Nacl and that was okay as well. Many thanks that I can now be off to the next hurdle! Raji On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam <[email protected]>wrote: > Hi Everyone, > > After several attempts to cleave the SUMO tag off my membrane protein > under various conditions (different reducing agents, enzyme-to-substrate > ratios, etc.) and after reading the manual and troubleshooting guide, I'm > reaching out to the ccp4bb community. > > Experiment: I dialyze the mixture of SUMO-membrane protein and Ulp1 > Express protease against buffer containing 20mM Tris pH 7, 150mM NaCl, 1mM > DTT or 2mM beta-mercaptoethanol and 0.02% DDM at 4C overnight (14-18 > hours). I am currently using an enzyme-to-substrate molar ratio of > 1-to-15-20. > > Problem: With buffer containing 1mM DTT, I get about 50% tag-cleaved > protein and 50% tagged protein. With buffer containing 2mM bME, I get about > 30% tag-cleaved protein and 70% tagged protein. > > Couple of things: > (1) Side-by-side cleavage of a SUMO-tagged control soluble protein by the > same batch of Ulp1 works to 100% completion. > (2) Detergent (0.02% DDM) did not interfere at all with cleavage of the > SUMO-tagged control soluble protein. > (3) I cannot set up the cleavage reaction at 30C or 37C and must stick > with 4C, a protocol that I have used successfully in the past with > SUMO-tagged soluble proteins. > > Although membrane proteins supposedly form a protein-detergent complex, I > wonder if some of my protein is in micelles and if the random orientation > of my SUMO-tagged protein in micelles may be the cause for incomplete > digestion. I've also suspected that some of my membrane protein may be > misfolded and oligomeric/aggregated, making the cleavage site inaccessible > to the protease. > > But suppose the above explanations are not the problem in my case and that > it's a technical issue and I am missing something very simple. Therefore, I > am planning to set up more reactions ramping up the ratio of > enzyme-to-substrate, increasing amount of bME (I prefer bME over DTT since > I need to rebind the cleaved mixture to His-affinity resin) and decreasing > the NaCl concentration to 100mM or lower (although 250mM NaCl did not > interfere with cleavage of control protein). > > Have folks working with SUMO-tagged membrane protein encountered similar > problems? I am purifying membrane protein from 30L bacterial culture and > the yields are not all that great. So, if possible, I'd like to get the > cleavage reaction to completion so that I don't have to suffer a 50% loss > of protein at this step. I have a construct for my membrane protein without > a SUMO tag and the expression is abysmal. > > Thanks very much for your time and suggestions! > Raji > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > >
