Re: [ccp4bb] frozen pellet insoluble protein
Andreas, you probably know all this, but I only understood quite recently. What happens is that as ice crystals form you get "brine rejection", the same thing that happens in the arctic when sea water freezes. Therefore you can have protein concentrated in pockets of high salt. Fine for some proteins, but others don't like it. And it can happen during (slow) thawing as well as during freezing. - Patrick On 29 September 2014 16:02, Andreas Förster wrote: > Dear all, > > I've encountered people who refuse to freeze cells and always lyse fresh > pellets. Better protein, they say. I've never had reason to do so myself, > or even to believe in their voodoo. Up until now, maybe. > > My protein expresses well and is almost all in the soluble fraction in an > expression test from a fresh pellet. The large-scale expression from the > same pellet, now frozen and thawed, yielded 90% insoluble protein. > > If it's the freezing that dooms the protein, I'm happy to redo the > fermentor run. Are there other examples out there of this? > > Thanks. > > > Andreas > > > > > -- > Andreas Förster > Crystallization and X-ray Facility Manager >Centre for Structural Biology > Imperial College London > -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36
Re: [ccp4bb] frozen pellet insoluble protein
Dear Andreas, maybe the problem is rather rooted in your expression set up. If the protein was soluble in the small scale test, it is not necessarily the case in a fermenter run. The parameters may have changed so strongly that your protein is now expressed mostly insoluble. To my experience, such things can even happen scaling up from small flasks to bigger ones. For some proteins it might be better to use fresh pellets. In my hands so far, I have not observed that freezing of unbroken cells matters, yet as I'm working on membrane proteins, I have noticed that as soon as the cells are broken and membranes are prepared, it can matter indeed if and for how long they are stored frozen. I guess, it needs to be tested for individual cases. Best wishes, Sabine On Mon, September 29, 2014 5:02 pm, Andreas Förster wrote: > Dear all, > > I've encountered people who refuse to freeze cells and always lyse fresh > pellets. Better protein, they say. I've never had reason to do so > myself, or even to believe in their voodoo. Up until now, maybe. > > My protein expresses well and is almost all in the soluble fraction in > an expression test from a fresh pellet. The large-scale expression from > the same pellet, now frozen and thawed, yielded 90% insoluble protein. > > If it's the freezing that dooms the protein, I'm happy to redo the > fermentor run. Are there other examples out there of this? > > Thanks. > > > Andreas > > > > > -- >Andreas Förster > Crystallization and X-ray Facility Manager > Centre for Structural Biology >Imperial College London >
Re: [ccp4bb] frozen pellet insoluble protein
I have experience with some proteins that don't tolerate freeze-thawing very well. It's hard to say exactly what the physical chemistry of this is, but it probably relates to (1) aggregation due to high concentration or protein or salts during the freezing process as water is removed, and/or (2) pH shifts due to changes in pKa of buffers/proteins as the temperature is lowered. Usually freezing in whole cells is less problematic than freezing purified protein solutions, but there are no absolutes. One protein we worked on could only be stabilized from cradle to grave in 20% glycerol, 100 mM DTT, and 4 deg C. Would not tolerate freezing, ever. Not even in cell pellets. Died at 25 deg C in a couple of hours--had to work quickly to do kinetics. Worst...protein...to work on...ever. Cheers, ___ Roger S. Rowlett Gordon & Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 9/29/2014 11:02 AM, Andreas Förster wrote: Dear all, I've encountered people who refuse to freeze cells and always lyse fresh pellets. Better protein, they say. I've never had reason to do so myself, or even to believe in their voodoo. Up until now, maybe. My protein expresses well and is almost all in the soluble fraction in an expression test from a fresh pellet. The large-scale expression from the same pellet, now frozen and thawed, yielded 90% insoluble protein. If it's the freezing that dooms the protein, I'm happy to redo the fermentor run. Are there other examples out there of this? Thanks. Andreas
[ccp4bb] frozen pellet insoluble protein
Dear all, I've encountered people who refuse to freeze cells and always lyse fresh pellets. Better protein, they say. I've never had reason to do so myself, or even to believe in their voodoo. Up until now, maybe. My protein expresses well and is almost all in the soluble fraction in an expression test from a fresh pellet. The large-scale expression from the same pellet, now frozen and thawed, yielded 90% insoluble protein. If it's the freezing that dooms the protein, I'm happy to redo the fermentor run. Are there other examples out there of this? Thanks. Andreas -- Andreas Förster Crystallization and X-ray Facility Manager Centre for Structural Biology Imperial College London
