Hi Venkat, I'll echo Nick and Pablo's points, especially the tendency of thrombin to cut off-target. I'll add that at least in my hands, Thrombin isn't a very stable protease, it's prone to self-digestion. Make sure you take care with it, flash freeze small aliquots and store them -80C. Many suppliers recommend storing in BSA, which will improve the stability but introduces a new protein contaminant to get rid of. Make sure you use only plastic vessels (especially if omitting BSA) as thrombin adheres to glass and you'll lose it. Lastly, if thrombin continues to be a problem, you can QuikChange in a PreScission or TEV site in place of the thrombin site - they tend to be much nicer proteases, and much more specific.
Hope this helps, Shane Caldwell McGill University On Wed, Jan 22, 2014 at 5:59 AM, Pablo Power <ppo...@ffyb.uba.ar> wrote: > Dear Venkat, > I totally agree with Nicholas in his appreciations. I would add that > sometimes thrombin cleavage could be a little bit "tricky", in the sense > that there are several secondary cleavage sites that are not always > specified in the brochures of your commercial thrombin. > You can search in google the full list of cleavage sites in order to look > if your protein could have them. But remember anyway: you MUST check your > digestion products by SDS-PAGE and compare it with your non-digested > protein to have an idea of what is happening. But I asume you did it, is > that right? > Try different conditions, move your Tag to the C-terminal (does your > protein have a signal peptide?), or simply avoid using HisTags if it result > in many problems. Use IEX, desalting and other columns; maybe more steps, > but it is probably a "safer" strategy. > > Kind regards, > > > > Pablo Power, PhD > Inv. Adjunto CONICET > Laboratorio de Resistencia Bacteriana > FFyB - UBA > Junin 956 (1113) - Buenos Aires, Argentina > Tel: +54 11 4964 8285 > Fax: +54 11 4508 3645 > > > 2014/1/22 Noinaj, Nicholas (NIH/NIDDK) [E] <noin...@niddk.nih.gov> > > Venkat, >> >> >> >> I haven't used thrombin for some time, but it doesn't sound like your >> choice of protease is your issue, assuming you are following the reaction >> conditions, etc. properly. So some general things I consider when having >> protease cleavage issues myself: >> >> >> >> 1- where are the extra 20 residues, before or after the thrombin site? >> According to the map for this vector, it only has 3 sites in the MCS >> region, with the max linker being 5-6 residues long. Regardless, I have >> had good luck adding one or more GlyGlyGly linker(s) immediately before my >> protein sequence. This can easily be done using site-directed mutagenesis. >> The problem is that, dependent on the protease and the folding of your >> protein target, the protease site may be partially or fully masked from the >> protease. >> >> >> >> You can also do a control where you heat denature your protein sample >> (only a small aliquot), cool, and then incubate with thrombin to see if you >> start to see any cleavage. >> >> >> >> 2- have you tried moving the HIStag/thrombin site to the C-term? again, >> the folding or oligomeric of your protein could lead to masking of the >> protease site and this might be resolved if the tag and thrombin site are >> on the C-term. >> >> >> >> 3- you mention that you tried thrombin cleavage once, only 1 time or >> several? if only 1 time, i suggest trying again, making sure to use the >> correct buffers, etc, and perform parallel reactions with increasing >> amounts of thrombin. If it still doesn't cut, then you will likely have to >> try other options with your tag and protease site at the construct/cloning >> stage. >> >> >> >> 4- lastly, many structures have been solved with the HIS tag still >> present, so i just wonder how many conditions have you screened already and >> how many buffers have you screened your protein in? While everyone has >> their favorites, i would start with my protein sample in either tris and >> HEPES buffers. Also, I suggest screening as many conditions as possible, >> we routinely screen 1000-1500 conditions for each new target. but of >> course, this would be dependent on how much sample you can produce and the >> resources available to you. >> >> >> >> >> >> Hope some of this helps, good luck with your sample and hope you work out >> your issues soon enough! >> >> >> >> >> >> >> >> >> >> >> >> Cheers, >> >> Nick >> >> >> >> >> >> >> >> -------------------------------- >> >> >> >> [ Nicholas Noinaj ] >> >> the Buchanan Lab >> >> Laboratory of Molecular Biology >> >> LMB-NIDDK, NIH >> >> 50 South Drive, Room 4505 >> >> Bethesda, MD 20892-8030 >> >> 1-301-594-9230 (lab) >> >> 1-859-893-4789 (cell) >> >> noin...@niddk.nih.gov >> >> >> >> [ the Buchanan Lab ] >> >> http://www-mslmb.niddk.nih.gov/buchanan/ >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> >> *From:* venkatareddy dadireddy [mailto:venkatda...@gmail.com] >> *Sent:* Wednesday, January 22, 2014 1:47 AM >> *To:* CCP4BB@JISCMAIL.AC.UK >> *Subject:* [ccp4bb] >> >> >> >> Dear All, >> >> >> >> My protein is cloned in pET-15b vector, contains His- tag, thrombin >> cleavage site and extra sequence of 20 amino acids from vector. I >> crystallized without cleaving extra sequence and never got any crystal >> hits/crystals. Once, I tried thrombin reaction and it didn't work. Here I >> would like to know how efficient the cleavage by thrombin and it is kind of >> you, can provide protocol and buffer system for reaction. >> >> >> >> Thank you >> >> Venkat. >> >> >> > >
