HELP!!!!!with protein renaturation (in a soluble form).

Scott McMahan mcmahan at oncology.wisc.edu
Wed Jun 12 23:12:11 EST 1996


In article <4pk103$dqk at taco.cc.ncsu.edu>, mrfaughn at unity.ncsu.edu (Mike
Faughn) wrote:

> Hello,
> 
>         The situation is this--  I would like to isolate plant proteins that
> interact with my His-tagged protein (of viral origin).  I am
> inoculating plants with the virus (which carries the His-tagged)
> protein and am hoping to use a NiNTA column to purify protein
> complexes.  Obviously I would like to do this under very gentle,
> native conditions.
> THE PROBLEM AT HAND--  Before spending tons of time trying to work out
> the native binding conditions for my His-tagged protein derived from
> infected plants I would like to "practice" with bacterially expressed
> protein.  I have expressed and purified the protein using the pET
> vector.  I have been using 8M urea and then slowly renaturing by
> dialysis.  The problem is that all the protein precipitates by the
> time I get down to a 200mM NaCl buffer.  This floculent stuff is no
> good for column work.  
> SO-- any ideas on how to get some soluble protein to "practice" with?
> 
> Thanksalot,
> mike 

I'm not sure what you mean by slowly renaturing by dialysis.  Do you mean
a step-wise decrease in the urea, ie dialyze into 4M then 2M then 1M etc? 
If not, you might want to try it.  Or you could try loading the denatured
protein onto the column in 8M urea and then washing the urea through
either going to 0M directly of stepwise as above.  Of course the not
reducing agent limitation on the column could lead to unwanted disulfide
formation.  A prep I've done with renatured inclusion bodies involved
dissolving them into 6M guanidine HCl and then diluting 2 fold in 6 steps
for a total 64 fold dilution.  I then batched it onto DE-52.  This was too
dilute when I tried the his-tagged version and Ni-NTA, though.

-- 
                                         Scott McMahan
                                         mcmahan at oncology.wisc.edu



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