Dear Raji, In our lab we always
(1) use TCEP (2) high molar ratio of protease with gentle rocking at 4C O/N (3) keep in mind the size (KDa) of the target protein regarding to the detergent micelle size. Although has been observed that in many cases the use of SUMO tags increases expression levels and the solubility of membrane proteins.It also has been observed that in some occasions SUMO tags (around 12KDa) are found to be inserted into the detergent micelle. e.g. SUMO tag (around 12KDa) + protein size (X KDa) should be > than the DDM micelle size (estimated size of 70KDa) 3C and SUMO proteases activities are greatly affected by the presence of detergent. You might try to increase a bit the linker or if your protein "allows" decrease the detergent just a little more (e.g 0.015% DDM) Good luck :) ------------------------------------------------------------ Dr Isabel De Moraes, MRSC Membrane Protein Laboratory Diamond Light Source ------------------------------------------------------------ On 16 Feb 2014, at 15:58, Raji Edayathumangalam 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 > -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
