HELP!!!!!with protein renaturation (in a soluble form).
qiagen at kaiwan.com
Tue Jun 18 08:50:59 EST 1996
On Tue, 11 Jun 1996, mrfaughn at unity.ncsu.edu (Mike Faughn) wrote:
>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?
There are many things which can affect renaturation of proteins, and a number
of papers on the subject. The ideal conditions vary from protein to
protein. Some key factors for difficult proteins seem to be the rate of
renaturation, and the presence of substances necessary for the protein to
remain soluble, such as cofactors, metal ions, pH conditions, or
detergents. The following paper may be of use to you:
Holzinger, A., Phillips, K.S. & Weaver, T.E. Single-step purification/
solubilization of recombinant proteins: Application to surfactant Protein
B. BioTechniques 20:804-808.
This paper describes how this precipitation problem can sometimes be solved.
The problem protein is left on the column while the buffer is changed from a
denaturing to a non-denaturing environment. This strategy maintains the
protein molecules in a spatially separated form which minimizes
aggregation and precipitation. The renatured protein is then eluted with 50
mM EDTA. In several of the examples described, the renatured protein stayed
If you need further help and advice on working with Ni-NTA resin, please call
QIAGEN Technical Service, or e-mail QIAGEN with your phone number, and
someone will contact you.
Temporary e-mail address: qiagen at Kaiwan.com
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