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 <mailto: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 <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 _____ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB <https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1> &A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
