By the way, I have an unrelated question. In the crystal structures containing yeast SUMO (including Ulp1-SMT1 complex: 1EUV), the first ~19 residues are absent. I am curious whether people tried a SUMO tag with these residues deleted. I am using the vector from invitrogen which seems to have the full-length ySUMO.
Thank you, Chen On Sun, Feb 16, 2014 at 10:38 PM, Matthew Bratkowski <[email protected]>wrote: > 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 > <[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 >> >> >
