[ccp4bb] Thrombin cleavage of membrane protein with fusion tag
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
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions in detergents. This is a rather extensive survey henceforth it is very informative. The variable detergent sensitivity of proteases that are utilized for recombinant protein affinity tag removal James M. Vergis 1, Michael C. Wiener in Protein Expression and Purification 78 (2011) 139–142 thrombin tends to be sensitive to reducing agents so I would stay away from DTT and TCEP , b-mercapto is acceptable in reasonable amounts. We cut in salt concentrations as high as 500 mM (NaCl) at pH 7.5-8.5 with 5-10% glycerol around no chelating agents should be present and imidazole in our hands tends to be an inhibitors (probably because it has some chelating/complexing activity). we have good cleavage in DDM , OG and somehow more difficulties in foscholines but it still cuts reasonnably well given the cost of the enzyme and the targets, the most crucial parameter is Protease/target ratio and incubation time and temp. you can do trials on small scale digests in PCR tubes at different temperatures. we usually cut at 4 or room temp. I hope this helps, Best regards, -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
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.eduwrote: 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 **
Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag
Dear Pascal and Toufic, Many thanks to both of you for the pointers. Toufic, I actually poked around online and bought exactly the same kit that you are suggesting, especially because the beads are reusable. So your experience is reassuring. So I'll find out soon. I also plan to play around with the detergent type and concentration among other things. I have no plans to take the shortcut but it's nice to have at least one other human being to talk to on these matters. I appreciate your responses :) Many thanks! Raji On Wed, Feb 20, 2013 at 1:15 PM, Toufic El Arnaout elarn...@tcd.ie wrote: 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.eduwrote: 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 ** -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University
