Hi Raji, I addition to the tips from Pascal, I would like to say that for a memb protein I worked on with a his-tag separated by a thrombin site, I used thrombin cross linked to agarose from Sigma (1 mL). The beads can be collected, washed and reequilibrated, making it ready for use so many times. In my case 1 mL of this thrombin was enough to cleave up to 5-10 mg of target protein, at 4 °C for 16 h (with slight rotation in a 15 mL tube, total volume up to 10 mL). In FC-12 there was a little better cleavage than in DDM. My opinion is that how thrombin (or most other proteases) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic
On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam <r...@brandeis.edu>wrote: > Hi Folks, > > Sorry this isn't a non-ccp4 post. > > I am working with a membrane protein for which I am finally able to scale > up expression. I am now also able to partially purify my protein from a > medium-scale (12-18L) bacterial culture using a two-step tandem affinity > purification protocol (Talon followed by amylose affinity steps). As the > next purification step, I am about to set up a pilot thrombin cleavage > experiment to separate my protein from the His.MBP fusion tag (see below). > > The construct that I am working with is as follows: > His.MBP--ThrombinSite--Membrane Protein > > There is only one theoretical thrombin cleavage site in the entire fusion > protein i.e., at the desired cleavage site with no theoretical secondary > sites. I would like to try cleavage both at 4C and around 25C from 4h to > overnight but I also have to balance the trials with the material I must > generate for the endless permutations and combinations one can try. Each > sensible pilot experiment is going to use up partially purified protein > from 6-12L preps. > > FYI. All purification buffers contain DDM and I haven't yet done extensive > detergent screens. > > Please could I ask the community to share tips/suggestions about > large-scale thrombin cleavage experiments with their favorite membrane > proteins. > > Many thanks. > Raji > > > -- > Raji Edayathumangalam > Instructor in Neurology, Harvard Medical School > Research Associate, Brigham and Women's Hospital > Visiting Research Scholar, Brandeis University > > -- ****************************************************** Toufic El Arnaout Trinity Biomedical Science Institute (TCD) 152-160 Pearse Street, Dublin 2 Tel.: +353 85 83 40 157 ******************************************************
