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<mailto: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.
