Hi all,
I have purified a protein from E. coli based system. All the purification steps were done without any ligand. However, the crystal structure of the protein showed a ligand density in the binding pocket that could be satisfied by placing the correct ligand which indicates that protein carries an endogenous ligand from the culture media. Now, we need apo-protein (ligand-free) to calculate the affinity between protein-ligand using either ITC or other techniques. Extensive dialysis in a ligand-free buffer (buffer, Sodium Chloride) did not work in our case.
Any suggestion is welcome to get the ligand-free protein.
Thank you
Shanti Pal
Dear Shanti
Theanswer is not obvoius,
it depends from the ligand or cofactor nature and interaction with the protein.
In some cases you can use a competitor:
eg
-If the ligand is a metal you can try to remove it by a chelating agent, as eg EDTA, and afterwards remove the complex edta-metal with a dialysis or desalting step.
- if the ligand bind charge aminoacid, eg Histidine, you can try to go at low ph, were it become protonated and less able to establish interactions.
if it is not possible and you are expressing your protein in E.coli you can try to replace your expression media with a chemical defined media which do not contain this cofactor. However also in this case the result is not obvious, because some cofactors are essential for the e.coli growth or can be synthetize from the e.coli itself also if not present into the media.
good luck
manuele
Dear Manuele,
Many thanks for the answer. The ligand is an amino acid, Serine. Therefore, low pH and chemical defined media probably would not work here. But I can try adding EDTA to the dialysis buffer.
Shanti Pal
Make sure that the ligand observed in the crystal structure is not a component of the crystallization medium, such as glycerol.
If the ligand is very tightly bound, but not covalent, then it may be possible to dislodge it by treating the protein with mildly denaturing conditions, such as 1 or 2 molar urea or sodium bromide, then remove the denaturant by dialysis. Ammonium sulfate precipitation, though not denaturing, may also work.
Thanks, Adam for the answer. I have tried mild denaturation followed by renaturation by removal of denaturant and it did not work. Either the protein did not denature well to remove the ligand or refolded well enough to bind the ligand in ITC.
However, I am curious to know how Ammonium sulfate precipitation would work to remove the tightly bound liagnd from the binding pocket?
Shanti Pal
To be honest, I'm not sure why ammonium sulfate precipitation works, but I know of 2 instances of it working on proteins I have studied. It may be related to the extremely high (but not denaturing) ionic strength of several molar ammonium sulfate. It may take a few cycles to get complete removal of the ligand. If there is a metal ion involved in binding of the ligand, then including EDTA would be worthwhile.
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