Re: [ccp4bb] Tev cleavage
You could try adding TEV to your protein just prior to crystallisation. I produced 2 equivalent structures this way; one with the tag clearly visible packing between molecules in an I centred space group and a 2nd (with TEV added) in P21 where the tag was no longer visible (the packing suggested it would not be accommodated and therefore cleaved). I don’t think many people try detagging their protein in conditions as extreme as those in a crystallisation trial but in the end that’s where the protein needs to be happy. Yes, there are lots of caveats with this technique, not least sub optimal cleavage conditions, but it’s a very simple experiment to do. Dave From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana Rangel Sent: 02 May 2015 18:57 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] Tev cleavage
Hi Giulliana, You can check our general protocol for TEV digestion (http://www.accelagen.com/TurboTEV-protocol.htm). Once you mixed TEV with protein solution, let it sit for digestion. No shaking or rocking. We found shaking or rocking sometimes reduce the efficiency. Some constructs just refuse to be cut by TEV. You may run analytical SEC or light scattering to see if your protein forms dimer or oligomers. The digestion is less or none for aggregated constructs. Find a buffer that your protein is most happy with. Some detergents work well with crystallization. TEV itself should be very stable in majority, if not all, of the buffers used for recombinant protein production. At least we never seen any issues with TurboTEV. As Karsten mentioned, HRV3C protease is more robust than TEV for tag cleavage. However HRV3C cut leaves 2 extra residues. Cheers, Chun From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana Rangel Sent: Saturday, May 2, 2015 10:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
Hey Giulliana, does your protein buffer contain high imidazole concentrations ( 150 mM)? If so you should try to exchange the buffer before cleavage, since the TEV protease tends to precipitate at higher imidazole concentrations. Schara Am 02.05.2015 um 19:56 schrieb Giulliana Rangel giulliana.ran...@gmail.com: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
Hi, I have used tev protease for tag cleavage during dialysis. In my case always tev got precipitated not my protein. And cleavage was always complete. I havE checked on the sds - page as wel. On 2 May 2015 23:27, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
[ccp4bb] Tev cleavage
Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
Hey, I always had very good results with cleaving with TEV at 4C overnight (30min are to short at this temperature). Alternatively, I did the TEV cleavage at 16-23C for 1-3 h. 37C might just be to warm for your protein... Since some time I switched to the PreScission system instead of using TEV protease. This enzyme works better in my hands than the TEV protease. If you need a really fast and efficient cleavage (15min / 4C), you can use a HIS-SUMO tag which can be cleaved off using ULP1. This is the most efficient cleavage in short time that I know. Good luck! Karsten Am 02.05.2015 um 10:56 schrieb Giulliana Rangel: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel -- Karsten Thierbach, Dr. rer. nat. California Institute of Technology Division of Chemistry Chemical Engineering Hoelz laboratory 1200 E. California Blvd., M/C 147-75 Pasadena, CA 91125, U.S.A.
Re: [ccp4bb] Tev cleavage
Giulliana, Alternatively to the previous excellent suggestions, you can add a few more residues between your protein and TEV-cleavage site. For example, if you have tag on the C-terminus you can add GSGS after the last residue: it should improve cleavage without significant impact on crystallization. Vitali From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK on behalf of Giulliana Rangel giulliana.ran...@gmail.com Sent: Saturday, May 2, 2015 12:56 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
Re: [ccp4bb] Tev cleavage
These are the usual culprits My buffers for cleavage and an on-column digestion worked good. (see below) Also most likely your TEV source (do not go cheap) enzyme is inactive (gone bad). Get a clone for TEV and make your own TEV in the lab. It save you a ton of money . 10 mM Tris-HCl (pH 8.0) 150 mM NaCl 0.1% IGEPAL CA-630 0.5 mM EDTA 1 mM DTT (add immediately before use from 1 M stock) Best wishes P On Sat, May 2, 2015 at 10:56 AM, Giulliana Rangel giulliana.ran...@gmail.com wrote: Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel -- P
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, We have had success setting up drops with TEV present. We simply added TEV at a 50:1 molar ratio and then set up the drops a couple of hours later. We went from having twinned crystals at 3A to untwinned at 2A, the crystal form also changed from orthorhombic to monoclinic, all in the same drop condition. We might have just got lucky, but it is an easy experiment to try. Cheers Peter ___ Dr Peter Czabotar Structural Biology Division The Walter and Eliza Hall Institute of Medical Research 1G Royal Parade, Parkville, Victoria, Australia Ph: (+61 3) 9345 2689 ___ On Apr 8, 2011, at 12:37 PM, anita p wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
Re: [ccp4bb] Tev Cleavage issue !!
Dear Anita, Sometimes the protein of interest has a relatively strong inherent binding affinity to the IMAC column. Have you tried to bind the cleavage reaction to an IMAC column and then elute using a shallow imidazole gradient? In fact, Porath developed IMAC chromatography as a tool for protein fractionation long before molecular biology provided the tools necessary to add poly-histidine tags to target proteins (Nature. 1975 Dec 18;258(5536):598-9). Best regards, Martin On Apr 8, 2011, at 4:37 AM, anita p wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could* highlight a bit more on this issue it would be helpful for me.* I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.comwrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, Admittedly, there are proteins that naturally bind to Ni-NTA so tightly that they co-elute with our His6-tagged proteins even on an imidazole gradient. However, we do need some luck to come across a protein with such property. For most proteins, they would just flow through the Ni-NTA in the presence of 10-20mM imidazole. Are you sure that what you saw as a lower molecular weight band on SDS gel was not really a clipped form of your protein that failed to be cleaved by TEV and still carried a His tag when undenatured? I guess that your TEV is also His-tagged, so seeing it in the Ni-NTA fraction is reasonable. But I would expect some sort of separation from your protein on the imidazole gradient even if the peaks are overlapping. Also, on Q column, TEV, being an extremely basic protein, simply won't bind. If you saw the TEV band in the Q fractions, then that suggests that you may have incorrectly identified your protein bands on the SDS gel. It would be interesting to see your SDS gel. Also providing more specific details of your chromatographies may help a lot. For example, what was the approximate concentration of imidazole when your peak came out from the Ni-NTA when eluted with a gradient? What was the condition you used for Q column and what is your protein's PI? There are just too many factors that could effect the performance of the ion-exchange chromatography. On the other hand, if it is true that your protein binds to Ni-NTA so well even without a His tag, then why not try expressing it alone without a His tag? Shouldn't you be able to purify it easily with Ni-NTA? Finally, the difficulty in TEV cleavage could indicate a construction problem. I assume that your protein is N-terminally His-tagged. To my experience, TEV wants one or two more amino acids between the G/S in ENLYFQ^G/S and the folded protein domain, i.e., it wants some space on the right hand side of the cleavage site. Adding one or two amino acids after the current cleavage site may help. Zhijie From: anita p Sent: Friday, April 08, 2011 5:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Tev Cleavage issue !! Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could highlight a bit more on this issue it would be helpful for me. I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.com wrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen
Re: [ccp4bb] Tev Cleavage issue !!
You could also try upping the tev:prot ratio, such that the protein is ~100% cleaved, then do IMAC or simply some other, non-IMAC chromatography step, such as ion exchange or SEC, depending on the size and charge of your protein relative to TEV. JPK On Fri, Apr 8, 2011 at 8:17 AM, Zhijie Li zhijie...@utoronto.ca wrote: Hi Anita, Admittedly, there are proteins that naturally bind to Ni-NTA so tightly that they co-elute with our His6-tagged proteins even on an imidazole gradient. However, we do need some luck to come across a protein with such property. For most proteins, they would just flow through the Ni-NTA in the presence of 10-20mM imidazole. Are you sure that what you saw as a lower molecular weight band on SDS gel was not really a clipped form of your protein that failed to be cleaved by TEV and still carried a His tag when undenatured? I guess that your TEV is also His-tagged, so seeing it in the Ni-NTA fraction is reasonable. But I would expect some sort of separation from your protein on the imidazole gradient even if the peaks are overlapping. Also, on Q column, TEV, being an extremely basic protein, simply won't bind. If you saw the TEV band in the Q fractions, then that suggests that you may have incorrectly identified your protein bands on the SDS gel. It would be interesting to see your SDS gel. Also providing more specific details of your chromatographies may help a lot. For example, what was the approximate concentration of imidazole when your peak came out from the Ni-NTA when eluted with a gradient? What was the condition you used for Q column and what is your protein's PI? There are just too many factors that could effect the performance of the ion-exchange chromatography. On the other hand, if it is true that your protein binds to Ni-NTA so well even without a His tag, then why not try expressing it alone without a His tag? Shouldn't you be able to purify it easily with Ni-NTA? Finally, the difficulty in TEV cleavage could indicate a construction problem. I assume that your protein is N-terminally His-tagged. To my experience, TEV wants one or two more amino acids between the G/S in ENLYFQ^G/S and the folded protein domain, i.e., it wants some space on the right hand side of the cleavage site. Adding one or two amino acids after the current cleavage site may help. Zhijie From: anita p Sent: Friday, April 08, 2011 5:10 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Tev Cleavage issue !! Thanks everyone for your suggestions ! Artem has pointed out that low diffraction of the crystal might be because of other problems .. If you could highlight a bit more on this issue it would be helpful for me. I have tried to seperate the cleaved, uncleaved and TEV over MonoQ column but there was a single peak and all of them came out together, there was no seperation. I even tried to cleave the protein at 30 degress and it starts precipitating. I have also tried binding it to the IMAC as Martin has suggested but then I get a single peak while running the imidazole gradient and its tev, cleaved and uncleaved together. And I also get the flowthrough while loading unto the column which should be theoritically the cleaved one but it is a combination of cleaved uncleaved and tev. awaiting for bit more suggestions Anita On Fri, Apr 8, 2011 at 10:48 AM, Artem Evdokimov artem.evdoki...@gmail.com wrote: For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run
Re: [ccp4bb] Tev Cleavage issue !!
I'd say since you obtained crystals with your tag it is not a disturbing factor and either disordered or making contacts. So removing the tag you might end up not getting crystals in the worst case. Now to the question why they don't diffract. Did you test the old fashioned way at RT in capillary ? Maybe your freezing is the problem. The inability to purify TEVed protein suggests that you have at least a dimer perhaps or higher multimer. Since you observe three bands on the gel, uncleaved, cleaved and TEV itself. Any idea about DLS or migration behavior on a SEC column for your uncleaved protein ? Since you have crystals what I would do is crush them up and rescreen whatever commercial screens you have available using some of the crushed crystals as seed. A second option would be to take your current condition and modify it with say the most frequent 12 cryo solutions added in maybe 5-10% effective concentration and see if you still obtain crystals. Before freezing them I would then freeze directly from the drop and a second crystal would be soaked into whatever concentration of the cryo is required to properly freeze. I bet you will find something that works. Of course you don't stop there. Once your crystals are on the gonio and diffract or don't diffract you will flash anneal them once or multiple times and report back in a nice table which condition resulted in the 2.3A dataset. So you have roughly 146 experiments to do alone from cryo- optimization. Good luck ! Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Hi Anita, so you tested your crystals inhouse, any idea how they do at the synchrotron ? Still no diffraction ? Since it's a hexamer I would expect the His-tag to be not so important and would rather rescreen with seeding first to see if any other conditions might result in diffracting crystals. Annealing did not help ? Can you slow down the growth of the crystals ? Another option you could try out is limited proteolysis and see if you get a stable fragment, then purify it via SEC and try it with the initial conditions but also rescreening. How big is your monomer ? Do you have a structural homolog / prediction ? Can you make better guesses what you should trim of by design ? Jürgen On Apr 8, 2011, at 11:43 PM, anita p wrote: Hi Jürgen I tried it by RT capillary as well as under liquid nitrogen, both dont diffract. I ran those on gel and then I confirmed that it is protein. On the SEC it runs as a hexamer.and the DLS shows that it is polydispersed. with regards Anita On Fri, Apr 8, 2011 at 10:03 PM, Jürgen Bosch jubo...@jhsph.edumailto:jubo...@jhsph.edu wrote: I'd say since you obtained crystals with your tag it is not a disturbing factor and either disordered or making contacts. So removing the tag you might end up not getting crystals in the worst case. Now to the question why they don't diffract. Did you test the old fashioned way at RT in capillary ? Maybe your freezing is the problem. The inability to purify TEVed protein suggests that you have at least a dimer perhaps or higher multimer. Since you observe three bands on the gel, uncleaved, cleaved and TEV itself. Any idea about DLS or migration behavior on a SEC column for your uncleaved protein ? Since you have crystals what I would do is crush them up and rescreen whatever commercial screens you have available using some of the crushed crystals as seed. A second option would be to take your current condition and modify it with say the most frequent 12 cryo solutions added in maybe 5-10% effective concentration and see if you still obtain crystals. Before freezing them I would then freeze directly from the drop and a second crystal would be soaked into whatever concentration of the cryo is required to properly freeze. I bet you will find something that works. Of course you don't stop there. Once your crystals are on the gonio and diffract or don't diffract you will flash anneal them once or multiple times and report back in a nice table which condition resulted in the 2.3A dataset. So you have roughly 146 experiments to do alone from cryo- optimization. Good luck ! .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742tel:%2B1-410-614-4742 Lab: +1-410-614-4894tel:%2B1-410-614-4894 Fax: +1-410-955-3655tel:%2B1-410-955-3655 http://web.mac.com/bosch_lab/ On Apr 7, 2011, at 22:37, anita p crystals...@gmail.commailto:crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. Is it probable that they dont diffract because of the extra his tag and the tev site? I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. Is there a possible way to approach this problem? Suggestions awaited Anita .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
[ccp4bb] Tev Cleavage issue !!
Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
For starters, you could re-clone the protein with e.g. just a His tag or move the tag to another end, or put some distance between the end of TEV site and the protein; or perhaps use no tag at all -- or a different one? Is it possible that the tag is messing you up - yes. Is it 'probable' - I can't say that I know because I've crystallized literally dozens of proteins with His-tags attached, and more than a few with His-tag and cleavage site. I prefer a plain His-tag or a cleaved one, to be sure - but I am not quick to blame the tag (since there are so many other possible things to blame). Based on the behavior of your protein after cleavage, it may be that you have oligomer(s) forming in solution such that cleaved and uncleaved proteins do not segregate. You may wish to explore other kinds of chromatographic separation e.g. ion exchange of HIC - they may or may not work out. You can also consider cleaving your protein at lower concentration, in the presence of detergents or polyols, etc. Cheers, Artem On Thu, Apr 7, 2011 at 9:37 PM, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
Re: [ccp4bb] Tev Cleavage issue !!
Hey Anita, I would like to add to Artem's comment that you can also try and cleave the protein at 30c for 2hr and then continue the cleavage overnight at 4c (you should check and see that your protein can withstand 30c incubation for 2hr, of course). In regard to your non-diffracting crystals - you can try seeding: Streak or Macro-seed your crystals onto a screen (screen seed) or onto the same conditions in which the crystals grew. Sometime you might get different morphologies that might diffract. Good luck, Chen --- Chen Guttman The Zarivach laboratory for Macromolecular Crystallography Building 39, Room 009B Ben-Gurion University of the Negev POBox 653 Zip Code 84105 Beer-Sheva Israel http://lifeserv.bgu.ac.il/wb/zarivach Tel. +972-8-6479519 Fax. +972-8-6472970 On Fri, Apr 8, 2011 at 04:37, anita p crystals...@gmail.com wrote: Hi Crystallographers, I am working of 23 Kda protein with a Nterminal His tag and a TEV cleavage site. I am getting crystals with the his tag and tev site intact, but they dont diffract. *Is it probable that they dont diffract because of the extra his tag and the tev site?* I am trying to get rid of this tag but the reaction is optimum at 10:1 protein to TEV ratio in micrograms overnight incubation without shaking. I tried to run it on histrap column after this reaction but I am not able to purify cleaved protein from TEV and uncleaved. I have tried several times but I get 3 bands ie., the TEV, uncleaved and Cleaved. I have also tried to use the Nibeads instead of the histrap column, but no difference is seen. * Is there a possible way to approach this problem?* Suggestions awaited Anita
[ccp4bb] TEV cleavage problems
Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF
Re: [ccp4bb] TEV cleavage problems
Hi Matthew, TEV is probably the least robust protease among those commonly used for tag removal. Here’s a common unit definition of TEV. One unit (corresponding to 0.1 ug TurboTEV) cleaves ≥85% of 3 μg control substrate in 1 hour at 30C. You need to use really a lot of TEV. Information collected from our customers shows large variation in quality of home-made TEV proteases. Some used 1:5 w:w ratio. Adding a little bit EDTA to Ni pool helps TEV digestion. You can find a general protocol at http://www.accelagen.com/TurboTEV-protocol.htm. Cheers, Chun Accelagen _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Matthew Merski Sent: Monday, May 24, 2010 9:28 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] TEV cleavage problems Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF
Re: [ccp4bb] TEV cleavage problems
Hi Matthew, TEV protease is very robust. I normally digest with 1:100 ratio according to the OD280. I normally digest at 4C for overnight around 16-18 hours. Make sure your tev protease site are not inaccessible and buried inside. best Xiaohu On Mon, May 24, 2010 at 12:27 PM, Matthew Merski mer...@blur.compbio.ucsf.edu wrote: Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF
Re: [ccp4bb] TEV cleavage problems
Ok, to sum up for the board, a good reference for this problem is at: http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf Thanks to everyone who responded. Matthew On Mon, May 24, 2010 at 9:27 AM, Matthew Merski mer...@blur.compbio.ucsf.edu wrote: Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF
Re: [ccp4bb] TEV cleavage problems
Hi Matthew, By now, you have received many posts telling you both how efficient and inefficient TEVp is. You might be confused. This seeming contradiction can be explained by a few events, among many others: Inaccessibility of cleavage site, absence of reducing agents, and presence of detergents if you are using them on membrane proteins. Solutions are, respectively: (1) redesign the construct to give the cleavage site some breathing room by adding a few residues around it, (2) add 2-mercaptoethanol, DTT, TCEP, etc. to keep TEVp active; it is a cysteine protease after all, (3) use a different detergent, enzyme, or ridiculously high amounts of enzyme (see Mohanty et al, Inhibition of tobacco etch virus protease activity by detergents, Protein Expression and Purification, 2002). It is true in my experience that TEVp is less active than thrombin and the like. But it is extremely specific, and much more than thrombin, so you don't have to worry about your protein being cleaved in unexpected places. Best, Engin On 5/24/10 12:43 PM, xiaohu mei wrote: Hi Matthew, TEV protease is very robust. I normally digest with 1:100 ratio according to the OD280. I normally digest at 4C for overnight around 16-18 hours. Make sure your tev protease site are not inaccessible and buried inside. best Xiaohu On Mon, May 24, 2010 at 12:27 PM, Matthew Merski mer...@blur.compbio.ucsf.edu mailto:mer...@blur.compbio.ucsf.edu wrote: Hello all, I am working with a protein that is expressed as with an N-terminal domain that is normally cleaved for activation of the protein (and crystallization). For in vitro reasons I've needed to switch the normal site to a TEV site. However, even though the TEV site is in the same place as the original proteolytic site, I have been unable to get cleavage despite using a lot of TEV at 37 C, pH 8.0. Has anyone been able to overcome a similar problem? Matthew Merski Post-doctoral researcher UCSF -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
