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 <[log in to unmask]> 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 <[log in to unmask]> 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