[ccp4bb] protein degradation
Hi i have setup a crystallization of a complex formed by two different proteins of molecular weights 53kD and 13kD. During purification of this complex there was slight degradation band of 53KD protein as observed from SDS PAGE but it did not effect complex formation. Crystals appeared after 8 months and on solving the structure i could find only 6kD fragment of the 53kD protein associated in complex form with 13kD protein and the rest of the fragment remains absent. Mass spec analysis with the crystals gave the same result. I couldn't explain this anomalous behavior. I have used different types of protease inhibitors and there is no autodegradation or autoproteolysis site. Can anyone please suggest what accounts for such unusual phenomenon. How to identify if any autoproteolysis event is taking place.
[ccp4bb] AW: [ccp4bb] protein degradation
Hi Supratim, if both the crystal and Mass spec tell you that you have 6k and 13k fragments, proteolysis definitively took place. There are several possible explanations for the phenomenom you observe: -time: processes that do not occur during the few hours of a normal biochemical experiments, may occur during a long (8 months!) crystallization experiments. In this time, also sites which are not official (auto)proteolysis sites may get cleaved. -concentration: proteolysis is usually a bimolecular event, which means that the reaction speed goes up with the square of the concentration. So when nothing may happen in a dilute solution, degradation can get very fast once you concentrate to crystallographic relevant (10 mg/ml) concentrations. -selection: if your fragments readily crystallize, but the parent protein does not, crystals will selectively accumulate the cleaved fragments. -contaminating protease: After purification, minute amounts of a contaminating protease may still be present, which over a long time in concentrated solutions, can wreak havoc with you protein. Also you may not have added the right inhibitor for this contaminating protease, or the inhibitor may get inactivated over time. The long time it took for your crystals to appear tells me that your parent protein does not want to crystallize and after 8 months, enough fragments had accumulated to crystallize instead. In your case, I would try the following: -add ligands to stabilize the protein and speed up crystallization -try different protein constructs. -try adding trypsin or chymotrypsin to do limited proteolysis during crystallization. Maybe you get larger fragments that also crystallize. -If you are really desperate: try antibodies or nanobodies. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von supratim dey Gesendet: Sonntag, 16. Juni 2013 15:22 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] protein degradation Hi i have setup a crystallization of a complex formed by two different proteins of molecular weights 53kD and 13kD. During purification of this complex there was slight degradation band of 53KD protein as observed from SDS PAGE but it did not effect complex formation. Crystals appeared after 8 months and on solving the structure i could find only 6kD fragment of the 53kD protein associated in complex form with 13kD protein and the rest of the fragment remains absent. Mass spec analysis with the crystals gave the same result. I couldn't explain this anomalous behavior. I have used different types of protease inhibitors and there is no autodegradation or autoproteolysis site. Can anyone please suggest what accounts for such unusual phenomenon. How to identify if any autoproteolysis event is taking place.
[ccp4bb] protein degradation in crystal
Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa
Re: [ccp4bb] protein degradation in crystal
Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.nowrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Were the crystals which run different on gels from the same drop, or separate drops? Yes, it is possible that the protein is being cleaved in the crystal (self-cleavage?); but it may also be that it is being cleaved in the mother liquor, and that crystallization is enriching one form or another. Remember that crystallization is a useful purification technique. There should be enough protein in a crystal to get some mass spec data, which will tell you the size of the components, and which should be precise enough to tell you the cleavage site. This will tell you what type of protease you are dealing with. Then as possible remedies: You could include an appropriate protease inhibitor during purification to limit degradation. You could add a bit of protease to encourage proteolysis to go to completion. You want your sample to be homogeneous, so whether this is useful depends on whether the cleaved part is the interesting part. You could engineer the gene with a stop codon at or near the cleavage site. This is what we did with HO-1, with some success. DOI: 10.1002/pro.5560070820 You could engineer the cleavage site to eliminate cleavage. Cheers, On 01/16/13 06:14, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several small bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] protein degradation in crystal
Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of John Domsic Sent: Wednesday, January 16, 2013 2:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.no wrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 tel:%2B47%202284%200794 http://www.jpmorth.dk
Re: [ccp4bb] protein degradation in crystal
Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex. Tom ** Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John Domsic *Sent:* Wednesday, January 16, 2013 2:22 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.nowrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 %2B47%202284%200794 http://www.jpmorth.dk -- Skype: tom.murray.rust Twitter: tmurrayrust http://twitpic.com/photos/tmurrayrust +44 7970 480 601 (UK)
Re: [ccp4bb] protein degradation in crystal
Could you identify the cleavage sites by protein sequencing and design new constructs (truncated versions) accordingly? It might improve your crystal quality to get better resolution. Joe On Wed, Jan 16, 2013 at 9:22 AM, Tom Murray-Rust tom.murray.r...@gmail.comwrote: Just to add to Herman's suggestions, if you are trying to crystallise a protease then you could also try using the S195A variant rather than an inhibitor. This would certainly be the case if you ever want to co-crystallise in a substrate, as PPACK (or the like) would occupy the active site cleft and prevent formation of the protease-substrate complex. Tom ** Hi John, This is really an amazing wild west story: the man who crystallizes faster than his protease! I really must compliment you with how you successfully performed these experiments! Unfortunately, proteins usually do not crystallize that fast (at least not in my hands), so in these cases other methods have to be used. As has mentioned before, protease inhibitors are the way to go. Especially with autolysis, as one protease cuts another one, the speed of the reaction goes with the square of the protease concentration. Whereas in dilute solutions not much happens, as soon as you start to concentrate towards crystallization conditions, say 10 mg/ml, degradation suddenly goes very fast. There are 2 cases to consider: 1) the protein you want to crystallize is a protease and is destroying itself. In this case you need to cocrystallize with a potent and specific inhibitor. With serine proteases, Wolfram Bode was very successful by using chloromethylketone-containing peptides (e.g. PPACK). These compounds would make covalent links with both the active site serine and histidine, effectively killing any protease activity. 2) the protein you want to crystallize is not a protease and it is a contaminant which is causing the problems. In this case I would add a protease inhibitor coctail in an earlier step of the purification to block the protease before the final purification steps. I would also add some small broad protease inhibitor e.g. PMSF to the protein solution used for crystallization. Herman -- *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *John Domsic *Sent:* Wednesday, January 16, 2013 2:22 PM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] protein degradation in crystal Hi Lisa, Speed is definitely a big factor here. With a protein I work with I can get large crystals in myriad conditions that only diffract to about 4-5 Ang. What I ended up doing was taking these crystals and seeding entire screens. I found that not only would crystals appear sooner but it revealed novel crystallization conditions. These seeded crystals would appear within minutes as Preben described and diffracted to better than 2 Ang. Another thought would be to try limited proteolysis to see if you can identify a more stable construct. -John -- John Domsic Postdoctoral Fellow Gene Expression and Regulation Program The Wistar Institute Philadelphia, PA 19104 On Wed, Jan 16, 2013 at 7:17 AM, jens Preben Morth j.p.mo...@ncmm.uio.no wrote: Dear Lisa It is not uncommon to see breakdown products when you run crystals on gel. Espesially if they are older crystals, sometimes you even see higher molecular bands, these are probably due to intra molecular cross links formed over time. If you are worried about stability, try to increase the crystallization speed, we have one example where we see a clear difference in both crystal quality and even space group depending on when we fish the crystals. The crystals appear within 5 min, the best quality data sets come from crystals we fish after only 30-60 min. You may also have a little protease contamination of course, to prevent this add protease inhibitor, or DTT, or EDTA to you protein before you set it up. cheers Preben On 1/16/13 12:14 PM, LISA wrote: Hi All, I have an 36KD protein which can be crystallize in two days. Most of the crystals are very big. But all cystals have poor resolution,lower than 3.8 A. I picked some crystals, washed them in the mother solution and then run SDS-PAGE. It is surprised to find that different cystals have different components. Some crystals have several samll bands below the band of the protein. And in some crysals the bigger size band (as the construct should be) almost disappared and have smear. Does the protein was degradated in the crystals? Did someone met the similar problem as I? Thanks All the best lisa -- J. Preben Morth, Ph.D Group Leader Membrane Transport Group Nordic EMBL Partnership Centre for Molecular Medicine Norway (NCMM) University of Oslo P.O.Box 1137 Blindern 0318 Oslo, Norway Email: j.p.mo...@ncmm.uio.no Tel: +47 2284 0794 %2B47%202284%200794 http://www.jpmorth.dk -- Skype: tom.murray.rust Twitter: tmurrayrust http
Re: [ccp4bb] Fwd: [ccp4bb] protein degradation
I agree with Mark, except that I wouldn't even try sonication, Triton or freeze/thaw cycles in that case. I'd look for emulsification (with a Homogenizer) in a cold room, but if you go quickly and gently with the French Press (either in a cold room or by using a cold piston) it might help. Don't use too much pressure, it heats up the sample. I also agree that if it migrates as a single peak in gel filtration and in heparin sepharose, there is no reason for not setting some drops with it. And if you decide to do it, then simplify the purification and avoid submitting the protein to treatments that are not helping to get it purer. (I just found it weird that your fraction 6 has a huge load of protein , I guess those are actually the beads from the purification or something like that? In any case it seems to me that the fraction volume could be increased) Carlos Em 15/02/2012, às 16:39, Mark J van Raaij escreveu: try experimenting with different, especially protease-deficient, E coli strains to express the protein and try different methods to lyse the bacteria (sonication, french-press, emulsification, bead-beater, mortar pestle under liquid nitrogen). on the other hand, if you are lucky, you are just proteolysing some surface loops and can still purify and crystallise the protein. This was done on purpose for the cap-binding complex, see: Crystal structure of the human nuclear cap binding complex. Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S. Mol Cell. 2001 Aug;8(2):383-96. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 15 Feb 2012, at 14:09, Sivasankar Putta wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta proteingel.pdf
Re: [ccp4bb] protein degradation
Late induction for short time. Then immediately purify it cut down on any unnecessary steps eg shorter spin all on ice or coldroom etc. 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 Feb 15, 2012, at 8:20, Sivasankar Putta sivasankarpu...@iisertvm.ac.inmailto:sivasankarpu...@iisertvm.ac.in wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta proteingel.pdf
Re: [ccp4bb] protein degradation
Hi, you may also check things like chemical degradation in SDS buffer as part of the analysis. Esspecially your degradation pattern is very much constant throughout your whole purification procedure. Christian Am Mittwoch 15 Februar 2012 14:09:19 schrieb Sivasankar Putta: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in* E. Coli.* The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta -- Christian Roth Institut für Bioanalytische Chemie Biotechnologisch-Biomedizinisches Zentrum Fakultät für Chemie und Mineralogie Universität Leipzig Deutscher Platz 5 04103 Leipzig Telefon: +49 (0)341 97 31316 Fax: +49 (0)341 97 31319
[ccp4bb] Fwd: [ccp4bb] protein degradation
try experimenting with different, especially protease-deficient, E coli strains to express the protein and try different methods to lyse the bacteria (sonication, french-press, emulsification, bead-beater, mortar pestle under liquid nitrogen). on the other hand, if you are lucky, you are just proteolysing some surface loops and can still purify and crystallise the protein. This was done on purpose for the cap-binding complex, see: Crystal structure of the human nuclear cap binding complex. Mazza C, Ohno M, Segref A, Mattaj IW, Cusack S. Mol Cell. 2001 Aug;8(2):383-96. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 15 Feb 2012, at 14:09, Sivasankar Putta wrote: Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta proteingel.pdf
Re: [ccp4bb] protein degradation
Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein. After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration). One small trick you can try is wash the cell with the buffer containing PMSF once before lysising the cell. Yu Xiaodi Date: Wed, 15 Feb 2012 18:39:19 +0530 From: sivasankarpu...@iisertvm.ac.in Subject: [ccp4bb] protein degradation To: CCP4BB@JISCMAIL.AC.UK Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta
Re: [ccp4bb] protein degradation
You can also try putting a different affinity tag on the other terminus, and use that as a second step. JPK On Wed, Feb 15, 2012 at 11:25 AM, Xiaodi Yu uppsala@hotmail.com wrote: Hi Sivasankar: Are you sure it is due to the protein degradation? Maybe you can try to do a western blot or others to check if it is the product of degradation. By the way, where you put the 6 histag, N- or C-terminal? If it is at the N terminal, maybe it is the truncation version of your protein. After looking at the gel, it seems your sample was over-load or had lots of unspecific binding to the column. Maybe you can add salt (250 mM NaCl, final concentration) and small amount of imidazle in the sample before you load onto the column (for example, 20 mM Imidazole final concentration). One small trick you can try is wash the cell with the buffer containing PMSF once before lysising the cell. Yu Xiaodi Date: Wed, 15 Feb 2012 18:39:19 +0530 From: sivasankarpu...@iisertvm.ac.in Subject: [ccp4bb] protein degradation To: CCP4BB@JISCMAIL.AC.UK Dear All, Can anybody suggest the tricks and trades of stabilizing a 133 kDa (multi domain) DNA binding protein, that we are expressing at 18 degree Centigrade in E. Coli. The protein appears to degrade during purification; we have protease inhibitor cocktail (in the lysis buffer) as well as 2 mM PMSF, 1 mM EDTA and 1mM DTT throughout during purification ( right from lysis stage). We handle the protein at 4 degree Centigrade. Can you please suggest what precautions we can try to avoid such degradation ? Please find the attached gel picture regarding protein Sivasankar Putta -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein degradation during concentration for crystallization trials
Hi, I've not tried this on column cleavage before, but have you tried first purifying the protein. cleaving the tag off the column and rerunning it through the column to capture the tag and washing off the protein? Also, with concentration. Going from a 50 mM buffer, and .3M salt, down to nearly water may not be great thing to do. Can your protein concentrate in your purification buffer to about .1-.2mM? If it can, I highly suggest trying to use the dialysis buttons from Hampton and screen a variety of different pHs, salts and additives to see what your protein can tolerate, before committing an entire purification prep to concentration. I've also noticed if you're using the centricon concentrators, make sure you take it out every few minutes and pipette it the solution a bit. These concentrators tend to create a concentration gradient, making the local concentration of protein very high in bottom of the concentrator, often times resulting in ppt. Hope it helps
[ccp4bb] protein degradation during concentration for crystallization trials
Dear all I am working on purification of 14 kd protein(pI 8.3, basic protein) that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the purification.I do on column cleavage with TEV to remove the tag (for crystallization purpose). Purification with MBP tag: After cleavage MBP also appear in elution fraction along with cleaved protein of my interest. To remove MBP(pI 5.0) from protein of my interest I have to do anion exchange chr. with DEAE resin(weak anion exchanger) with buffer of pH7.3. Hence I do dialysis against the buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room. I have few problems: 1) why the MBP elute with protein of my interest ( I tried at low salt concentration also but still elute). 2) During dialysis my protein of interest precipitate. 3) If I tried FPLC, protein of interest and MBP elute in same fraction with superdex 200 column. Problem with GST tag purification: it give me impure protein even after enough washes with high salt concentration buffer (upto 2 molar). When i concentrate protein in CENTRICON (Millipore, centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer with 5mM BME) it degrade very fast and probably aggregate as smear obtain on SDS page below the size of protein after concentration (even protease inhibitor not very much helpful) and band intensity of protein of interest almost remain same before and after concentration step. Please send me your valuable suggestion to overcome to these difficulty. I have also tried with some additives such as sucrose, glycerol, PBS buffer. With regards -- $$ VIKRANT Junior Research fellow Cancer Research Institute Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, India $$$ Send free SMS to your Friends on Mobile from your Yahoo! Messenger. Download Now! http://messenger.yahoo.com/download.php
Re: [ccp4bb] protein degradation during concentration for crystallization trials
Hi, MBP tag: 1.there might be a TEV cleavage site in your MBP variant. 2. your protein needs salt to stay in solution 3. your protein forms aggregates with MBP GST tag: you probably concentrate a protease together with your protein. You need a protease inhibitor kit to take care of different types of proteases. It looks like a His tag would be a better option for you. Maia vikrant saa wrote: Dear all I am working on purification of 14 kd protein(pI 8.3, basic protein) that has MBP(maltose binding protein, 45 kd,) tag, and same protein in other vector(pGEX-KT) that has GST tag. During affinity purification in both cases I used 300mM nacl and 50 mM tris, pH 7.5 buffer throughout the purification.I do on column cleavage with TEV to remove the tag (for crystallization purpose). Purification with MBP tag: After cleavage MBP also appear in elution fraction along with cleaved protein of my interest. To remove MBP(pI 5.0) from protein of my interest I have to do anion exchange chr. with DEAE resin(weak anion exchanger) with buffer of pH7.3. Hence I do dialysis against the buffer 20mM nacl and 20 mM tris, ph 7.3. in cold room. I have few problems: 1) why the MBP elute with protein of my interest ( I tried at low salt concentration also but still elute). 2) During dialysis my protein of interest precipitate. 3) If I tried FPLC, protein of interest and MBP elute in same fraction with superdex 200 column. Problem with GST tag purification: it give me impure protein even after enough washes with high salt concentration buffer (upto 2 molar). When i concentrate protein in CENTRICON (Millipore, centrifugation 4000rpm at 4 degree, 300mM NaCl and 50 mm Tris buffer with 5mM BME) it degrade very fast and probably aggregate as smear obtain on SDS page below the size of protein after concentration (even protease inhibitor not very much helpful) and band intensity of protein of interest almost remain same before and after concentration step. Please send me your valuable suggestion to overcome to these difficulty. I have also tried with some additives such as sucrose, glycerol, PBS buffer. With regards -- $$ VIKRANT Junior Research fellow Cancer Research Institute Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Kharghar, Navi Mumbai, India $$$ ** ** Send free SMS to your Friends on Mobile from your Yahoo! Messenger. Download Now! http://messenger.yahoo.com/download.php
Re: [ccp4bb] protein degradation
Hi Debajyoti, There is another simple thing you can try: Raise your NaCl concentration to 500 or 1000 mM in your lysis buffer. This helps to clean up your sample further and it might inhibit proteases in your lysate. Good luck, christian Debajyoti Dutta wrote: Hi, This is going to be an off topic question concerning this community. I have a protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole found to be degraded, appears like a deep band with other bands (touching each other below the main band) in SDS PAGE. The protein is a DNA binding (pI~ 10) and Tris-Hcl buffer is used with 10% glycerol and 300mM NaCl. for purification. Does Phosphate buffer do any help in stopping the degradation. All suggestions are welcome. Thank you for your reply in advance. Sincerely Debajyoti Dutta Rediff Shopping http://adworks.rediff.com/cgi-bin/AdWorks/click.cgi/www.rediff.com/signature-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3OAS_QUERY=null ___ Dr. Christian Biertümpfel Laboratory of Molecular Biology NIDDK/National Institutes of Health phone: +1 301 402 4647 9000 Rockville Pike, Bldg. 5, Rm. B1-03 fax: +1 301 496 0201 Bethesda, MD 20892-0580 USA ___
Re: [ccp4bb] protein degradation
If your protein precipitates at low ionic strength you may want to establish what concentration of salt does it tolerate (by slowly diluting a fraction of your pool with water just until you see precipitation) then try to adjust your cation exchange conditions so that you load with enough salt to keep the protein happy, then elute with higher salt concentration. You may want to adjust the pH of your buffer to be lower - usually this helps keep things in solution with basic proteins such as yours. In general you don't *have* to dialyze prior to ion exchange - dilution works just as well. Imidazole and histidine are pretty much the same in terms of protein behavior, at least in my experience. Both of them can cause 'apparent' protein degradation - if you take a sample of your protein with histidine or imidazole still in it, then boil it with the gel loading buffer you can get fake proteolysis (i.e. only in the boiled sample). It does not happen with every protein, but it does happen (some weird chemistry involving the imidazole ring at high temp. no doubt). Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Debajyoti Dutta Sent: Sunday, August 24, 2008 3:21 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein degradation Hi again, Thank you for all you have replied. I suspect the sonnication for such a bad result. I am just wondering if I can use Histidine instead of Imidazole and then buffer exchange to go for Cation exchanger. Does Imidazole had any bad effect in protein. I have experienced that the protein cannot sustain low salt and get ppt during dialysis. Sincerely Debajyoti Dutta On Sat, 23 Aug 2008 Artem Evdokimov wrote : Hi, If you are plagued by 'generic' proteolysis, it is not likely that changing buffers from TRIS to phosphate will help reduce the breakdown. You may want to ask yourself several key questions regarding the breakdown: 1. is it proteolysis or abortive translation? 2. is it happening during expression or post-lysis? 3. is it exoproteolysis or endo- (or both)? Which terminus is more affected? If you have abortive translation or alternate transcription/translation starts, you should examine the DNA/RNA sequence of your gene closely - sometimes you can tell (e.g. beginning of the gene is peppered with methionines and S-D sequences) If you have proteolysis during expression, it sometimes can be alleviated or even eliminated by changing expression conditions (temperature and richness of media seem to be key - this summer we had exactly this kind of a situation where expression at 37C for 4 hours gave more total protein than expression at 20C overnight, however the 37C protein was significantly 'busted up' whereas the 20C was essentially intact). If you have proteolysis during cell lysis you may be able to reduce its extent by means of at least the following (not a complete list by any means!): a) lyse and process the cells on ice or even in liquid nitrogen (see some previous posts regarding mortar-and-pestle LN2 lysis). Avoid sonication or other forms of mechanical lysis in-liquid as they all generate heat (detergent lysis or French press may be safe alternatives) b) make an effort to lyse the cells and complete primary extraction as fast as possible (for example, in an extreme case you can forego the lysate clarification step - high-flow IMAC resins can tolerate complete crude lysate in batch mode or sometimes even on a column). c) use protease inhibitors (can be somewhat expensive) If you can figure out which end of the protein is affected (or perhaps its lysis of an interdomain linker, exposed loop, etc.) you may be able to re-design the construct to avoid this. Presumably the fact that your protein can be extracted via the His6 tag means that the end with the tag is intact (or at least enough of it is intact to make purification possible). Therefore it may be interesting to switch the tag to the other end - you may get lucky. If you cannot afford the (expensive) commercial protease inhibitor cocktails, you may be able to get away with using the 'old basics' such as benzamidine, PMSF (AEBSF), and EDTA/EGTA or phenantroline. Yes, you won't be able to use IMAC together with EDTA - but you can always do cation exchange first (protein with pI of 10!!!) in EDTA, then polish it up with IMAC etc. If your protein is really pI 10, it should bind to CM-sepharose in buffer with pH as high as 8, which is essentially a guarrantee that your protein will be almost alone in the extracted material - you wil likely see a ribosomal p10 protein that's about pI 11 - and that's it. Remember that if you go this way, you should avoid using lysozyme for cell lysis since HEWL is mighty basic itself. Good luck, Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Debajyoti Dutta Sent: Friday, August 22, 2008 6:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein
[ccp4bb] protein degradation
Hi, This is going to be an off topic question concerning this community. I have a protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole found to be degraded, appears like a deep band with other bands (touching each other below the main band) in SDS PAGE. The protein is a DNA binding (pI~ 10) and Tris-Hcl buffer is used with 10% glycerol and 300mM NaCl. for purification. Does Phosphate buffer do any help in stopping the degradation. All suggestions are welcome. Thank you for your reply in advance. Sincerely Debajyoti Dutta
Re: [ccp4bb] protein degradation
Hi, If you are plagued by 'generic' proteolysis, it is not likely that changing buffers from TRIS to phosphate will help reduce the breakdown. You may want to ask yourself several key questions regarding the breakdown: 1. is it proteolysis or abortive translation? 2. is it happening during expression or post-lysis? 3. is it exoproteolysis or endo- (or both)? Which terminus is more affected? If you have abortive translation or alternate transcription/translation starts, you should examine the DNA/RNA sequence of your gene closely - sometimes you can tell (e.g. beginning of the gene is peppered with methionines and S-D sequences) If you have proteolysis during expression, it sometimes can be alleviated or even eliminated by changing expression conditions (temperature and richness of media seem to be key - this summer we had exactly this kind of a situation where expression at 37C for 4 hours gave more total protein than expression at 20C overnight, however the 37C protein was significantly 'busted up' whereas the 20C was essentially intact). If you have proteolysis during cell lysis you may be able to reduce its extent by means of at least the following (not a complete list by any means!): a) lyse and process the cells on ice or even in liquid nitrogen (see some previous posts regarding mortar-and-pestle LN2 lysis). Avoid sonication or other forms of mechanical lysis in-liquid as they all generate heat (detergent lysis or French press may be safe alternatives) b) make an effort to lyse the cells and complete primary extraction as fast as possible (for example, in an extreme case you can forego the lysate clarification step - high-flow IMAC resins can tolerate complete crude lysate in batch mode or sometimes even on a column). c) use protease inhibitors (can be somewhat expensive) If you can figure out which end of the protein is affected (or perhaps its lysis of an interdomain linker, exposed loop, etc.) you may be able to re-design the construct to avoid this. Presumably the fact that your protein can be extracted via the His6 tag means that the end with the tag is intact (or at least enough of it is intact to make purification possible). Therefore it may be interesting to switch the tag to the other end - you may get lucky. If you cannot afford the (expensive) commercial protease inhibitor cocktails, you may be able to get away with using the 'old basics' such as benzamidine, PMSF (AEBSF), and EDTA/EGTA or phenantroline. Yes, you won't be able to use IMAC together with EDTA - but you can always do cation exchange first (protein with pI of 10!!!) in EDTA, then polish it up with IMAC etc. If your protein is really pI 10, it should bind to CM-sepharose in buffer with pH as high as 8, which is essentially a guarrantee that your protein will be almost alone in the extracted material - you wil likely see a ribosomal p10 protein that's about pI 11 - and that's it. Remember that if you go this way, you should avoid using lysozyme for cell lysis since HEWL is mighty basic itself. Good luck, Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Debajyoti Dutta Sent: Friday, August 22, 2008 6:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein degradation Hi, This is going to be an off topic question concerning this community. I have a protein 6XHis tagged. When retrieved from the Ni-NTA column with imidazole found to be degraded, appears like a deep band with other bands (touching each other below the main band) in SDS PAGE. The protein is a DNA binding (pI~ 10) and Tris-Hcl buffer is used with 10% glycerol and 300mM NaCl. for purification. Does Phosphate buffer do any help in stopping the degradation. All suggestions are welcome. Thank you for your reply in advance. Sincerely Debajyoti Dutta http://adworks.rediff.com/cgi-bin/AdWorks/click.cgi/www.rediff.com/signatur e-home.htm/[EMAIL PROTECTED]/2206641_2199021/2201650/1?PARTNER=3OAS_QUERY= null Rediff Shopping
Re: [ccp4bb] protein degradation?
Hi, if I recall this correctly, it is the nickel that is in your sample after elution and boiling your protein in SDS sample buffer does the rest. So, could be the sample is fully okay!!! J. Tiago Botelho wrote: Hi, I also had a similar problem with one of my proteins... I had it cloned in two different plasmids, one with Cter His-tag and the other in the Nter. Whenever I purified it using IMAC purification I would get the double band (that I confirmed by MS and were the same). I got ride of this double band when I decided to simply avoid Ni-IMAC purification and just use ion-exchange methods like Q/S Shepharose. It seems that I had not degradation but simply some chemical alteration due to the use of IMAC column. Good luck and best regards, Tiago. --- Tiago Botelho PhD Student IBMB - CSIC Institut de Biologia Molecular de Barcelona carrer Jordi Girona 18-26, 08034 Barcelona Phone: +34 93 4006100 ext. 269/332 Fax: +34 93 2045904 www.ibmb.csic.es On Mon, November 5, 2007 4:01 am, Juergen Bosch wrote: Another possibility, since you say MS looks identical and you are unable to separate those two bands by other chromatografic means, is simple a metal binding site in your protein. If the charge is changed in your protein due to metal binding then the apparent molecular weight will differ - that then could explain the identical results in MS (if I understand what you say regarding the MS correctly, if the masses are different, then forget about my sentences) Try incubating your protein with e.g. EDTA and see if you get a single band in your SDS PAGE. Jürgen Vijay Kumar wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay -- 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
[ccp4bb] protein degradation?
Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay
Re: [ccp4bb] protein degradation?
Hi Vijay, If it is C-terminal degradation, fusing the His-tag to the C-terminus may help you get rid of it. Wataru Kagawa # Wataru Kagawa, Ph. D. Research Scientist Protein Research Group RIKEN (Physical and Chemical Research Institute) W221, West Research Bldg. 1-7-22 Suehiro-cho, Tsurumi-ku Yokohama, Japan 230-0045 tel.045-503-9206 fax.045-503-9201 #
Re: [ccp4bb] protein degradation?
Are you expressing a eukaryotic protein? If so, you might want to check for rare codons. There are a number of websites where you can put in your coding sequence and check. I recently had this issue and it turned out to be incomplete/stalled translation rather than proteolysis as I had several rare codons in succession. You can circumvent this by adding an antibiotic selectable plasmids encoding the rare tRNAs. Good luck Eric On 11/4/07, Vijay Kumar [EMAIL PROTECTED] wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED]
[ccp4bb] 答复: [ccp4bb] protein degradation?
Hi Vijay, It may be caused by the redox status of your proteins, which is normal in redox related proteins, especially caused by cysteine residues. The upper band may be the reduced form and the lower oxidized form, which can be found in numerous papers. Addition of oxidants or reductants in purification may solve the question and homogenize your proteins, regardless of the result of SDS-PAGE. First of all, check the redox status of your proteins. Best Regards and Good Luck! Sincerely yours, Jiang Yu School of Life Sciences University of Science and Technology of China _ 发件人: CCP4 bulletin board [mailto:[EMAIL PROTECTED] 代表 Vijay Kumar 发送时间: 2007年11月4日 20:22 收件人: CCP4BB@JISCMAIL.AC.UK 主题: [ccp4bb] protein degradation? Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay
Re: [ccp4bb] protein degradation?
On Nov 4, 2007, at 14:23, Eric Dollins wrote: Are you expressing a eukaryotic protein? If so, you might want to check for rare codons. There are a number of websites where you can put in your coding sequence and check. I recently had this issue and it turned out to be incomplete/stalled translation rather than proteolysis as I had several rare codons in succession. You can circumvent this by adding an antibiotic selectable plasmids encoding the rare tRNAs. indeed thats often the case; there are commercial cells with plasmids encoding for rare codons, rosetta-2 work really well for us. A. Good luck Eric On 11/4/07, Vijay Kumar [EMAIL PROTECTED] wrote: Hi, I have been trying purify a N-ter his-tagged protein over- expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay -- D. Eric Dollins, Ph.D. C266 LSRC, Research Dr. Duke University Medical Center Durham, NC 27710 (919) 681-1668, [EMAIL PROTECTED]
Re: [ccp4bb] protein degradation?
Vijay, It is hard to guess what you mean when you say that 'mass spec results confirmed both protein bands are the same'. Do you mean that both bands were cut, digested with e.g. trypsin, and the resulting peptides were mapped? Or, alternatively, you've done an MS spectrum on the sample that went on the gel, and found only one species? If it is the first case then the two bands are not necessarily (chemically) 'the same' because peptide mapping is almost never complete, and certain types of PTM cannot be detected like that. Therefore you should perform the other experiment (i.e. direct MS of the sample using a high-resolution ESI-MS instrument) and see what protein species are present in the sample. If it is the second case, i.e. you've already done the direct mass determination and found no obvious signs that there are two species present, then the situation is slightly more challenging. The interpretation of MS results depends on the quality of the spectrum both in terms of signal/noise ratio and in terms of the resolution of the instrument. It also depends on the size of the protein - typically larger proteins are more difficult and additional chemical species can slip by undetected. If your experiment results are confident to within a few daltons, then you probably have a 'subtle' event such as deamidation of an amide, resulting in an acid side chain (and an additional charge), a redox state change (S-S bond is only 2 Da different from SH SH pair), or a conformational change that persists even in SDS-PAGE (some more stubborn proteins can remain fully or partially folded even in SDS). Also, you can have a strong metal binding which would persist through SDS-PAGE but may be dislodged by TFA, sinapinic acid, etc. during the MS experiment. There are other unstable modifications that may be noticeable on the gel but completely (and artefactually) destroyed by the MS. Likewise, there are gel artefacts e.g. associated with DP clipping at higher temperatures, that may not appear on MS. If you only did e.g. MALDI-TOF kind of experiment, then your resolution may be low enough (especially for larger proteins) that changes in one to a few amino acids may slip by undetected. Clearly, the solution here is to do the other kind of MS, to see if at higher resolution things don't look more interesting. Hope this helps, Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Vijay Kumar Sent: Sunday, November 04, 2007 7:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein degradation? Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay
Re: [ccp4bb] protein degradation?
Some proteases are metal-dependent, and inhibitors for those aren't Ni-column-compatible. We (meaning my students) found that it helps to (1) work very quickly and (2) put EDTA into the fraction collector tubes before eluting from the Ni column. At 06:22 AM 11/4/2007, Vijay Kumar wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay --- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] protein degradation?
Yes! And if you have money, you can use phosphoramidon http://delphi.phys.univ-tours.fr/Prolysis/phosphoramidon.html It's nearly as good as EDTA or phenantroline for inhibiting metalloproteases - and it's fully compatible with IMAC! Artem -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of [EMAIL PROTECTED] Sent: Sunday, November 04, 2007 5:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] protein degradation? Some proteases are metal-dependent, and inhibitors for those aren't Ni-column-compatible. We (meaning my students) found that it helps to (1) work very quickly and (2) put EDTA into the fraction collector tubes before eluting from the Ni column. At 06:22 AM 11/4/2007, Vijay Kumar wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay --- Phoebe A. Rice Assoc. Prof., Dept. of Biochemistry Molecular Biology The University of Chicago phone 773 834 1723 fax 773 702 0439 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alpha betically.php?faculty_id=123 http://www.nasa.gov/mission_pages/cassini/multimedia/pia06064.html
Re: [ccp4bb] protein degradation?
Another possibility, since you say MS looks identical and you are unable to separate those two bands by other chromatografic means, is simple a metal binding site in your protein. If the charge is changed in your protein due to metal binding then the apparent molecular weight will differ - that then could explain the identical results in MS (if I understand what you say regarding the MS correctly, if the masses are different, then forget about my sentences) Try incubating your protein with e.g. EDTA and see if you get a single band in your SDS PAGE. Jürgen Vijay Kumar wrote: Hi, I have been trying purify a N-ter his-tagged protein over-expressed in E.coli. After purification (Ni-NTA or Co-Talon), I find two bands in SDS PAGE which are very close each other (top band in the right MW and more intense than the lower band). Western blot (for his-tag) of the gel gave signal for both the bands. Mass spec results confirmed both protein bands are the same. So I think it could be C-ter degradation of my protein. Also the 2 bands exist after ion-exchange and sizing column. I use commercially available complete protease inhibitor tablets (increasing concentration has no effect) and sonication for lysis. I am wondering if people have encountered the same problem and got any suggestions? Thanks in advance. Regards, Vijay -- 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
