Re: [ccp4bb] off topic: problematic protein
I would like to thank all of you who promptly replied to my posting with so many ideas and suggestions (18 answers so far). I will post a summary soon. best wishes to all Savvas On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] off topic: problematic protein
I would think thing here is that this protein actually associates to those lipid nanodiscs...(around the disc) and Na cholate CMC is around 10 mM. so, yes you can solubilise proteins that bind lipids, the question is does this protein bind lipids or not? or is it just scrambled or whatever, doesnt like to be overexpressed etc??? or sticky because it is part of a larger complex naturally and not stable alone, for instance. which i wouldn't know of course. regards, Tommi On Apr 20, 2011, at 1:08 AM, Arthur Glasfeld wrote: I recently followed a protocol from Stephen Sligar's lab for the purification of his nanodisc protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I saw stuff coming off the column in both washes. The reference is: Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856. http://pubs.acs.org/doi/abs/10.1021/nl025623k Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non- micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/ tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
[ccp4bb] off topic: problematic protein
Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] off topic: problematic protein
I recently followed a protocol from Stephen Sligar's lab for the purification of his nanodisc protein, which has strong hydrophobic character as it associates with phospholipids. His protocol includes washes with 1% Triton X-100 and then with 50 mM cholate (both at pH 8 in the presence of 300 mM NaCl). Worked great, and I saw stuff coming off the column in both washes. The reference is: Bayburt et al. (2002) Nano Letters, vol. 2, pp 853-856. http://pubs.acs.org/doi/abs/10.1021/nl025623k Good luck, Arthur Arthur Glasfeld Department of Chemistry Reed College 3203 SE Woodstock Blvd. Portland, OR 97202 USA On Apr 19, 2011, at 12:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
Re: [ccp4bb] off topic: problematic protein
HI Savvas, We recently had a protein that showed two overlapping peaks on the disposable fast flow Q columns, so we decided to see if we could resolve them with a higher resolution Q media. It ended up having 7 distinct peaks, only one of which was free of contaminants. We have also noticed that the presence of heat shock protein bound to our favorite protein is highly dependent on the induction time/temp, and also varies between bacterial strains. It is also effected by the media. Yo might try osmotic shock or other additives in the media. We have also had luck by adding 1-3 molar urea in the lysis buffer. We get lots more protein out, with better purity, but some of it crashes, which I think is purifying out the misfolded protein. Lastly, you might try a fusion protein to something that has chaperone activity, like MBP, which may mask the binding epitopes for the other proteins. Best regards, Kendall Nettles On Apr 19, 2011, at 3:48 PM, Savvas Savvides wrote: Dear colleagues We are working on a large bacterial protein (featuring a large number of repeats) that appears to copurify with a lot of other proteins after Ni-affinity chromatography and gel-filtration. We have tried adjusting the ionic strength of these runs and have gone to as high as 5M NaCl but only saw marginal improvements. It appears that the protein likes to stick to a lot of stuff, and in fact the number of repeats in a given construct appears to correlate with the extent of contaminants in our purification steps. We have admittedly never seen anything like this among the so many different, and often challenging, proteins, we have worked on in our group over the last few years. We are now thinking of trying detergents in the buffers (at non-micellar concentrations), in conjunction with playing a bit with the pH to see if such an approach provides a 'stripping' effect. Interestingly, the protein has a calculated pI of 3.5 ! As the options for handling this protein are indeed quite numerous, we would be grateful for any additional input and possible tips/tricks. I will prompty post a summary of the thread. Best regards Savvas et al. Savvas Savvides Unit for Structural Biology @ L-ProBE Ghent University K.L. Ledeganckstraat 35, 9000 Ghent, Belgium Tel/SMS/texting +32 (0)472 928 519 Skype: savvas.savvides_skype http://www.LProBE.ugent.be/xray.html
