Have you tried maintaining the protein in a solution with a low concentration of excess cofactor? Light vs dark? Excess reducing agent or different reducing agent or oxygen-depleted and under argon?
John > On Feb 2, 2020, at 4:22 AM, Jon Hughes <[email protected]> > 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 <[email protected]> Im Auftrag von Artem > Evdokimov > Gesendet: Sonntag, 2. Februar 2020 00:46 > An: [email protected] > 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 <[email protected]> > 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
