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what makes a protein insoluble? - (Apr/27/2006 )

as my urea PAGE post has been forgotten rolleyes.gif ...im trying to make it work but its not working mad.gif ...im asking myself what would make my protein insoluble? i mean why would it aggregate? i know that instead of foliding into the native form it folds not properly and forms aggregates in ex E-coli differnt medium and no chaperones etc etc....but what i am interested to know (biochemists!!) for example any specific structure would lead to its insolubility (more beta sheet, helix ??) like structurally how will it become insoluble?? huh.gif ...am I talking total nonsense here? laugh.gif

-Kathy-

QUOTE (Kathy @ Apr 28 2006, 02:31 PM)
as my urea PAGE post has been forgotten rolleyes.gif ...im trying to make it work but its not working mad.gif ...im asking myself what would make my protein insoluble? i mean why would it aggregate? i know that instead of foliding into the native form it folds not properly and forms aggregates in ex E-coli differnt medium and no chaperones etc etc....but what i am interested to know (biochemists!!) for example any specific structure would lead to its insolubility (more beta sheet, helix ??) like structurally how will it become insoluble?? huh.gif ...am I talking total nonsense here? laugh.gif

Hi.

No, you're not talking nonsense. The number of papers that have been written about the problem of insuloble proteins would make your head spin! Try pubmed and see how many hits you get!!!
To answer your question: lots and lots and lots of things can make proteins insoluble in E.coli (which is your problem, I think). The top choices include induction temperature, [IPTG], protein size in some cases, cell strain, codon bias (sometimes the ribosome gets part-way through synthesising, runs out of tRNAs, and sits down for a break; meanwhile the whole system backs up, and hey presto, you have an inclusion body especially if your sequence has long runs of the same residue), and protein sequence, if there are problems elsewhere.
I understand that the biggest problems come when you try to make the cell produce too much protein too quickly. Slow things down (by dropping the temperature or [IPTG] for example) and your cells may be much happier.

-swanny-

swanny, thank you for the reply! actually i want to reduce the temperature and IPTG and timing and i'll do it someday rolleyes.gif ...but as I got so much of pure protein from the inclusion body im tempted to purify it... biggrin.gif ...but as I decrease Urea concentration it precipitates...and when I try urea/glycerol PAGE it also precipitates.... dry.gif ....so im trying to figure out what to do.... wacko.gif

-Kathy-

QUOTE (Kathy @ Apr 28 2006, 05:30 PM)
swanny, thank you for the reply! actually i want to reduce the temperature and IPTG and timing and i'll do it someday rolleyes.gif ...but as I got so much of pure protein from the inclusion body im tempted to purify it... biggrin.gif ...but as I decrease Urea concentration it precipitates...and when I try urea/glycerol PAGE it also precipitates.... dry.gif ....so im trying to figure out what to do.... wacko.gif


It really depends on what you want to do with your protein. If you are planning on some functional studies, I'd try to be as gentle as you can be. If you just want to digest it to work with fragments, you can be rough with it. I would suggest you try slowing down the expression before anything else: it might have no effect on your protein yield, and it might just give you lots of soluble, folded protein. You will never know until you do the experiment (which is why we are scientists, isn't it?)

You might want to consider the total protein concentration. Your protein might have a low saturation concentration, above which it wants to self-associate. Try the dialysis at different starting concentrations, and see if you still get precipitation.

Unfortunately, you might just have a protein that you need to keep at low concentration, and/or with urea or other chaotropic salts added. Do you have any luck with high salts in your system? I mean, does the dialysis to remove urea work better if you have say 0.5 M NaCl?

-swanny-