[ccp4bb] Purification with ligand

[protein.chemist protein.chemist]

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

Re: [ccp4bb] Purification with ligand

[Pascal Egea]

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

Re: [ccp4bb] Purification with ligand

[Artem Evdokimov]

Hi,

 

I wouldn't call this a standard procedure - generally speaking the most
important two parameters you have to consider are: the protein/ligand
affinity and the supply of the ligand. For tightly bound ligands, you may be
able to just add the ligand once (i.e. during cell growth and expression, or
upon lysis) thus avoiding the need to add the ligand to the chromatographic
buffers throughout the process. This is perfect for ligands that aren't
available in large quantities. The flip side of this is that when you're
done purifying you're kind of stuck with the ligand you have - unless you
successfully exchange it for something else (which is often hard to do
completely if ligand has single digint nanomolar Ki or lower). On the other
end of the spectrum are ligands that bind not as tightly and can be readily
exchanged - these require you to have the ligand everywhere in the
chromatography in the amount sufficient to elicit nearly full occupancy of
the active site. Obviously it's better if you have such ligands in good
supply.

 

Examples of the tight-ligand co-purification are: GPCRs, nuclear hormone
receptors (PPAR/RXR, RAR/RXR, etc.) - these proteins often require ligands
for stability; and certain kinases that are otherwise toxic to the cells
(PKC family) and have to be expressed/purified with a ligand because
otherwise they kill the expression host.

 

Examples of the medium- or weakly- bound ligand co-purifications include
many nucleotide/side acting enzymes, aminotransferases (often need PLP to
remain active and stable), proteins that require specific metal ions to
remain in good shape, and a number of proteases that tend to chew themselves
up unless they have an inhibitor to gnaw on.

 

Sometimes ligands are co-purified unintentionally (famous example - quorum
sensing factor with the crazy boron chemical) and pop up in the electron
density as a surprise to the crystallographer.

 

Please don't hesitate to ask specific questions :-)

 

Artem

 

---

When the Weasel comes to give New Year's greetings to the Chickens no good
intentions are in his mind.

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
protein.chemist protein.chemist
Sent: Thursday, February 26, 2009 5:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Purification with ligand

 

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