Sorry for my typo, it is Ulp1-SMT3 complex...
On Sun, Feb 16, 2014 at 10:54 PM, Chen Zhao <c.z...@yale.edu> wrote: > 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 > <mab...@cornell.edu>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 <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 >>> >>> >> >
