Hi Raji- To echo what John Lee said, we found that we had to maintain a ratio between 1:2 and 1:10 Ulp1:substrate for several different target proteins, none of which were membrane proteins however. So, I would definitely try maybe a 1:5 ratio and see what happens. This wasn't too cost prohibitive as we were expressing a His-tagged Ulp1 and could afford to use quite a bit when we needed to. We also found that having 5-10% glycerol and 0.2% NP-40 (Igepal) enhances cleavage and helped keep our newly cleaved target protein from precipitating. Most importantly, we discovered that we could not simply scale up the volume of the cleavage reaction after running small volume pilot experiments. So, we would set up numerous 10 mL cleavage reactions in 15 mL conical tubes, incubate, and every 30 minutes or so we would invert the tubes to mix (we were mainly incubating at 30 C for ~3 hours). Doing it this way we found we went from ~50% cleavage to ~80-90%. I realize that duplicating that is impractical in your case but perhaps you could set up an orbital or shaker at 4C and gently keep the solution moving for the incubation. By the way, we tested dialysis and on-column cleavage and neither was better than the above method in our hands. Happy to discuss further or answer any questions off board.
Best- Brad On Mon, Feb 17, 2014 at 10:50 AM, Raji Edayathumangalam <[email protected]>wrote: > Hi Folks, > > Thanks very much for your helpful suggestions. Several folks have > suggested switching to TCEP. I am also going to increase the molar ratio > of Ulp1 in the cleavage reaction like John Lee has suggested since I did > not think of the effect of DDM on Ulp1. (Everything seemed to work in the > case of the control SUMO-tagged soluble protein in presence of DDM so I > didn't think of that as an issue at the time.) If these tweaks do not end > up working in my case, I'm inclined to make and test a construct with a > longer linker region, like several people have suggested. > > Many thanks again! > 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 >> >> > > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > >
