Hi Mariah,
You may need to specify what type of ligand (is it a nucleotide, a
small synthetic molecule, a peptide etc ?) and also what is the affinity
between your ligand and your protein.
I have purified several protein-ligand complexes, you can go several routes.
If you have a high affinity binder and fairly 'cheap' or abundant ligand it
is possible for example to add it in your crude extract (or even in your
bacterial culture in some extreme cases). It may end-up improving your
'expression yield' in terms of soluble protein readily available in your
initial extract.
I have used this approach successfully when purifying the
ligand-binding domain of a nuclear receptor with its very hydrophobic
natural ligand (retinoic acid) or a synthetic drug. In this case it was
giving a more homogenous population of protein at the start of the
purification. I included the hormone in the crude extract after sonication
and centrifugation (so at an early stage of the purification). After that I
kept ligand around in the buffer (Cobalt affinity chromatography and gel
filtration (at low concentration) and kept adding ligand to the concentrated
protein. If your ligand has some kind of specific UV absorption, it can be
very easy to monitor its presence and the 'saturation level' of your
protein. If you have a high-affinity binder, it can be a very efficient way
to start with homogenous population of protein-ligand complex.
This approach is really useful when your ligand happens to be only soluble
in protein-unfriendly solvents like ethanol or acetone (this was the case
for retinoic acid); in the crude extract despite the addition of alcohol,
your protein won't suffer too much from the presence of added alcohol and if
affinity is high and you add enough of it, you will efficiently saturate it.
In another case, a complex between two GTPases, I had to use a
non-hydrolyzable GTP analog. The compound was far too expensive to be used
in the crude extract. In this case , we purified the two apo-proteins
separately, formed the complex in presence of ligand and included ligand in
the ion-exchange chromatography buffers and in the gel filtration buffer (at
a low concentration though but it helped us to stabilize the complex). Again
it all depends on the affinity.If you decide to include ligand in your
gel-filtration buffer, keep also in mind that you will contaminate your
columns and it can be hard to get rid of some ligands sometimes.
Sorry, if all this was a little bit too long.
Hope this helps,
Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics
Laboratory of Robert Stroud
On Thu, Feb 26, 2009 at 2:58 PM, protein.chemist protein.chemist <
pp73...@gmail.com> wrote:
> Hello,
> I wanted to know if there is a standard procedure for purification of
> protein with ligand. I have never done this before so it will be nice to
> get some help.
>
> Thanks,
> Mariah
>
> --
> Mariah Jones
> Department of Biochemistry
> University of Florida
>
