[ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
On Nov 11, 2008, at 16:12, Tim Gruene wrote: Hi Jorge, using a system where you can cleave off the His-tag (with TEV, FactorX, etc) adds a purification step which is complementary to the first purification step (Ni-column etc). In my experience this results in very pure protein which makes it more likely to crystallise. 3C protease seems to work really well and would recommend it over TEV or FX. One could look at the His-SUMO tag ideas for alternative idea for cleavage and some aid in solubilization. Therefore I would always choose such a system and not spend much time on trying to crystallise the protein with the His-tag attached. If you have the His-protein always try to crystalize it. We got one very nice structure with a His-zipper from the Tag helping to form the lattice. Consider the no-tag as yet another alternative for crystallization trials - but if that does not work cleave it ! A. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Chris, Can you or others speculate, perhaps in light of the structure, why a his-tag would cause precipitation? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Christopher Bahl" <[EMAIL PROTECTED]> To: Sent: Tuesday, November 11, 2008 10:06 AM Subject: Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags Hi Jorge, We have had cases both for and against leaving His-tags on proteins. In one case, the presence of the His-tag was causing the protein to precipitate, blocking our attempts at crystallization until we removed it. However, we also had a different protein that crystallized beautifully with the His-tag left on. I can't speak to the impact the tag had on crystallization since we never bothered to remove it and it was not visible in the structure. My (rather unhelpful) input is that it greatly depends on the individual protein as to whether or not it is beneficial to remove the His-tag prior to crystallization. -Chris Tim Gruene wrote: Hi Jorge, using a system where you can cleave off the His-tag (with TEV, FactorX, etc) adds a purification step which is complementary to the first purification step (Ni-column etc). In my experience this results in very pure protein which makes it more likely to crystallise. Therefore I would always choose such a system and not spend much time on trying to crystallise the protein with the His-tag attached. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
I seem to recall a paper by the SGC group showing their flowscheme, all proteins are first crystallized with the His-tagged variant, only if no crystals are obtained or they diffract poorly a TEV cleaved version is purified. I think the success rate of solved structures between His-tagged and non-tagged was in the range of 70%. Sure one could argue if they had cleaved off the tag to begin with and compared both batches at the same time the ratio of His to non-His might be different. Nevertheless it demonstrates that you don't need to cleave of a His tag to be successful in obtaining a structure. If I should find the paper again I will post the reference. Jürgen On 11 Nov 2008, at 06:34, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Hi Jorge, using a system where you can cleave off the His-tag (with TEV, FactorX, etc) adds a purification step which is complementary to the first purification step (Ni-column etc). In my experience this results in very pure protein which makes it more likely to crystallise. Therefore I would always choose such a system and not spend much time on trying to crystallise the protein with the His-tag attached. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Another theory is that trace amounts of Ni may leech off the column during purification and coordinate with multiple His-tags on the pure protein, causing them to aggregate. For sure. We had a case where a protein would precipitate after post-Ni-NTA dialysis. The precipitate would go back into solution if resuspended in the presence of imidazole or EDTA/EGTA. Inclusion of a bit of EDTA into fractions and dialysis solution completely eliminated precipitation. Dima
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Hi Jorge, We have had cases both for and against leaving His-tags on proteins. In one case, the presence of the His-tag was causing the protein to precipitate, blocking our attempts at crystallization until we removed it. However, we also had a different protein that crystallized beautifully with the His-tag left on. I can't speak to the impact the tag had on crystallization since we never bothered to remove it and it was not visible in the structure. My (rather unhelpful) input is that it greatly depends on the individual protein as to whether or not it is beneficial to remove the His-tag prior to crystallization. -Chris Tim Gruene wrote: Hi Jorge, using a system where you can cleave off the His-tag (with TEV, FactorX, etc) adds a purification step which is complementary to the first purification step (Ni-column etc). In my experience this results in very pure protein which makes it more likely to crystallise. Therefore I would always choose such a system and not spend much time on trying to crystallise the protein with the His-tag attached. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
In our case, incubation in the presence of imidazole can knock the protein out of solution- this was gleaned from crystallization conditions containing imidazole. The best we could come up with is that the imidazole groups from the His-tag were interacting with neighboring protein in solution, causing them to precipitate. Another theory is that trace amounts of Ni may leech off the column during purification and coordinate with multiple His-tags on the pure protein, causing them to aggregate. Hope that helps, -Chris Jacob Keller wrote: Chris, Can you or others speculate, perhaps in light of the structure, why a his-tag would cause precipitation? Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program Dallos Laboratory F. Searle 1-240 2240 Campus Drive Evanston IL 60208 lab: 847.491.2438 cel: 773.608.9185 email: [EMAIL PROTECTED] *** - Original Message - From: "Christopher Bahl" <[EMAIL PROTECTED]> To: Sent: Tuesday, November 11, 2008 10:06 AM Subject: Re: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags Hi Jorge, We have had cases both for and against leaving His-tags on proteins. In one case, the presence of the His-tag was causing the protein to precipitate, blocking our attempts at crystallization until we removed it. However, we also had a different protein that crystallized beautifully with the His-tag left on. I can't speak to the impact the tag had on crystallization since we never bothered to remove it and it was not visible in the structure. My (rather unhelpful) input is that it greatly depends on the individual protein as to whether or not it is beneficial to remove the His-tag prior to crystallization. -Chris Tim Gruene wrote: Hi Jorge, using a system where you can cleave off the His-tag (with TEV, FactorX, etc) adds a purification step which is complementary to the first purification step (Ni-column etc). In my experience this results in very pure protein which makes it more likely to crystallise. Therefore I would always choose such a system and not spend much time on trying to crystallise the protein with the His-tag attached. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 11 Nov 2008, [EMAIL PROTECTED] wrote: Dear all, Concerning the crystallization of proteins with a His-tag, based upon discussions on this bulletin board and on the article Mike Carson, David H. Johnson, Heather McDonald, Christie Brouillette and Lawrence J. DeLucas, His-tag impact on structure, Acta Cryst. D63, 295-301, (2007). I understand that as a general approach one should try to crystallize the protein with the His-tag (yet it might crystallize, so no need to care the work of taking out the tag); then, if this is not successful, go to the procedure to either express (and purify) it without the His tag or take it out later. Any observations/advices? But one other question to add is what if the protein is expressed with both a c-myc tag and a His-tag (you might consider also the case in which only the c-myc tag is present). Any experience on the effect of the c-myc tag on crystallization? References are welcome yet I could not find much googling around... Thanks, Jorge
[ccp4bb] Fwd: [ccp4bb] crystallization of proteins with His-tag and/or c-myc tags
Returning to the previous discussion, we've had luck crystallizing several proteins with a His-tag. We've used both N-terminal and C-terminal his tags, and have had success with both of them. In one of our structures, the his tag actually formed an interface for crystal packing. We have also tried using the "Tagzyme" kit to remove the tags of our proteins that were not crystallizing. We found that cleavage of the His-tag with Tagzyme was very unsuccessful for our proteins. Kathleen
