Hi Raji, I have no experience with membrane proteins, but I have used SUMO tags frequently. Unlike other proteases that cleave at a specific site (thrombin, TEV, etc.), Ulp1 recognizes the tertiary structure of SUMO for cleavage. So if only about 50% of your protein is cleaved, this may indicate that about 50% of your protein is misfolded. You may just try to take the cleaved protein and use it and forget about recovering the uncleaved portion. While your yield will obviously be substantially reduced, you only really want correctly folded protein for structural or functional studies, and the inability of Ulp1 to cleave the SUMO tag could serve as means of removing misfolded protein from your sample.
Matt On Sun, Feb 16, 2014 at 9:58 AM, Raji Edayathumangalam <r...@brandeis.edu>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 > >
