Does somebody purified only the polyQ domain of a protein? Only the polyQ domain without more regions of the protein or without fusioning any globular protein behind.
Thank you so much!
Hi Cristina,
I'm working for many years with polyQ polypeptides by following their aggregation kinetics, in collaboration with Fabrice Klein.
The main trouble with polyQ expression is the aggregation occuring during the different steps.
The idea is to express and purify a fusion protein (MBP-PolyQ) containing a cleavage site between the carrier protein and the PolyQ. The carrier protein is used for affinity purification, and is highly useful to avoid aggregation over the expression and purification steps. The last purification step is done by size exclusion chromatography, leading to a pure and monomeric MBP-PolyQ.
As soon as you want to start your experiment with the PolyQ itself, you just need to add the protease specific to your cleavage site to release your PolyQ polypeptide. This step enables you to synchronize your polyQ regarding to aggregation processes.
For more details, you can give a look to the paper Davranche et al. Human Molecular Genetics (2011), Vol 20, n 14.
All my best.
Yves
That's great information!! Thank you so much for this detail answer! I will take a look to your reccomendations and start designing a plasmid!
Best regards and thanks again!
Dear Yves,
now it is time ago that I asked for this question.
I have a GST-polyQ domain and I have the problem that when I express it, the fusion protein is cut inside the cell. So, after purification, I have very few amount of fused protein.
Did you have this problem? How can I avoid it?
I tried different strains and different expression conditions and there is no way to have a nice expression band with the correspondent MW and also to have good expression levels.
Thank you so much,
Cristina
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