Thank you for the clarification! First of all, many proteins will exhibit this sort of behavior when stored in liquid form at moderately low temperature.
One solution is to store in glycerol (35 percent and up) at -20C. Or in deep freeze at -80C or in LN2. The one remaining question that would be useful to address is the degree of homogeneity in your sample. Is 100% of your protein folded and functional? We have seen cases where a small population of poorly folded protein would give rise to aggregates that eventually seed the entire sample into aggregation. Removing these seeds early in the game was key to keeping the whole sample happy. In a few very curious cases we were able to achieve this by controlled heating (e.g. 55C for 5 minutes) of the sample followed by ultracentrifugation or some other separative technique. The idea is to accelerate aggregation of the busted protein and then to crash the aggregate out. This is of course somewhat extreme and cannot be recommended as a general solution to every issue of this sort. Addition of the right detergent can also help. Separation via another orthogonal matrix is often helpful as well. Artem On Sun, Feb 2, 2020, 4:22 AM Jon Hughes <jon.hug...@bot3.bio.uni-giessen.de> wrote: > Thanks for you interest. Ok, here are some more details. > > The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa) > holophytochrome, produced with a C-terminal His6 tag together with its > co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red > photochromic (that is, photoactive) such that, as a 2 component sensory > histidine autokinase / phosphotransferase, its kinase activity can be > switched on and off by appropriate light pulses. Thus it is unambiguously > functional. It is also highly soluble (10 mg/ml is no problem) – but > subsequently (over days and weeks) it aggregates (irrespective of the > photostate) to form a fluffy precipitate. > > Incidentally, I believe that most SHPK's and indeed most phytochromes have > aggregation problems like this. > > Beyond urea being a less potent chaotrope than guanidinium/HCl, the > different chemical actions of the two might give a hint as to what causes > the aggregation. > > Cheers > > jon > > > > *Von:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> *Im Auftrag von *Artem > Evdokimov > *Gesendet:* Sonntag, 2. Februar 2020 00:46 > *An:* CCP4BB@JISCMAIL.AC.UK > *Betreff:* Re: [ccp4bb] Urea vs. Guanidinium/HCl > > > > More details would be helpful. Do you know whether your protein is folded > and active to begin with? Many partially folded proteins behave in a way > that resembles your experience... Urea is a less potent denaturant mole for > mole than GuHCl so it is not super surprising that it behaves differently. > > > > Artem > > > > On Sat, Feb 1, 2020, 6:22 PM Jon Hughes < > jon.hug...@bot3.bio.uni-giessen.de> wrote: > > Hello everyone, > We work on a protein that tends to aggregate. The process is slowed but not > stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl > dissolves the aggregate readily, urea just turns it into an amorphous > chewing-gum-like mass. Does that info provide anyone with a clue as to why > the aggregation occurs and maybe suggest how to stop it in a way that would > not thwart crystal formation? > Best, > jon > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
