Protein precipitating need help choosing suitable buffering system - (Mar/21/2011 )

I'm currently trying to purify proteins that have a pI of ~9.3-9.8. I'm trying to use a two-step purification protoccol, first by Nickel affinity chromatography followed by size exclusion using an FPLC machine. My lysis buffer is 8M Urea, 0.1M NaH2PO4, 0.01M Tris, 1M KCl (pH 8). My protoccol then calls to buffer exchange whilst the proteins are bound to the beads in a step wise process and eventually elute in 450mM KCl, 50mM HEPES, 200mM Imidazole (pH 7.5). This seems to cause precipitation of some of the proteins onto the beads as they can no longer be regenerated fully. The eluted protein is dialysed overnight into 450mM KCl, 50mM HEPES to remove the imidazole and if left for extended periods the next day, starts forming aggregates. Nevertheless, enough protein survives to undergo size exclusion separation using the same buffer. However, once the protein comes out of the column, it can no longer be concentrated down to usable levels. As it precipitates if concentrated more than around 2X. (Precipitation due to dilution is a real phenomenon right?)
I would greatly appreciate your help if you have any suggestions to reduce the the precipitation of my protein so that i can concentrate it once purified. The problem is, the protein then needs to be used with live cells so i'm trying to avoid additives to the buffer such as reducing agents and detergents which might be toxic, also i'd like to avoid using more salt for the same reasons. Making the above buffer with 10% glycerol did not help. Do you think maybe i should use a different buffering system to HEPES? Should i change the pH of my buffer considering the pI of the proteins? Thankyou in advance for any suggestions and feel free to e-mail me
Thanks!

Hola, it could be better if you don´t use urea in the lysis buffer and you will improve solubility if work at pH far away of pI as 7-6.5. If you sonicate or freeze and tawing 3 times each, and centrifugue , if your protein or a part of it was in the supernatant , after filtration you could load the 6His resin and your results could be improved. Buena suerte

Thanks for your help protolder,I was wondering whether you have a bit of an explanation as to why removing urea would improve solubility? I thought it did the opposite. Do you think it's because the solution is so saturated already? The reason i use urea is that my protein becomes part of inclusion bodies when expressed and becomes insoluble and urea is there to put it back into solution. Even then, after spinning, a significant amount is still in the insoluble pellet which cannot be retrieved with 8M urea. What are your thoughts?

protolder on Mon Mar 21 10:34:24 2011 said:

glikal on Tue Mar 22 06:29:26 2011 said:

Hola, sorry obviously urea improves solubility. I wanted to say that if you have a little part of soluble protein withouth urea it will be easier purify it ,than a process of desnaturalization/refolding, because, or your protein is simple or refolding could recover only a little part of it well folded. Well, I would work in expression with a light induction or withouth it (if there is any constitutive one). With an strain wich facilitates folding of your protein for instance any Origami or Rosetta-gami strains, lactose induced at 25ºC,short harvesting times and lysis/sonication buffer phospate pH 6 .If all this efforts don´t give any part of your protein soluble your protocol is correct only you have to low pH. Buena suerte