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
