I am in a fix... One of the reviewer asked us to prove that disassociated subunit (namely beta 5) of human 20S proteasome is catalytic active. Any suggestion will be helpful. All that comes to my mind is use a mild detergent treated 20S proteasome and further purify (affinity chromatography?) beta5 subunit. please help
the purification of the beta 5 subunit in low to quantitative (mg) amounts could be achieved by free flow electrohoresis. We already have experiance in purifying 20S proteasome, see linked Publication.
http://www.ncbi.nlm.nih.gov/pubmed/17660509
Hi Adam, you might also be able to overexpress a tagged version of the beta5 subunit. In that case, you could do a much more rigorous purification using the affinity tag on the beta5 subunit.. I am assuming you want to show activity of the separate subunit in the absence of total proteasome? On the other hand, there are also several activity-based probes that can bind active subunits, and can then be visualized on SDS-PAGE. Here is an overview. http://www.ncbi.nlm.nih.gov/pubmed/23700161
That seems rather like topic for subsequent paper to me, but OK.
My suggestion (but I have no experience with proteasome, so really only suggestion):
first try conditions that will dissociate proteasome, yet keep the activity. You should see shift of activity peak on gel permeation chromatography after treatment.
Second, you can either proceed directly with the fractions from gelpermeation chromatography, to analyse which peptides are in active fractions, but that has low resolution and I think the subunits have similar sizes, right? In that case you can try another, some unspecific chromatogrpahy like ionex to separate your proteins and check which one is the active one.
With this set-up you will not need to clone your subunit again, include any taggs etc.
Tomas fully agree It sound like another paper. We will try our best but initial study with detergents are totally underwhelming. Detergent at low concentration activates proteasome but it is intact (fully 20S) further increasing of detergent results in activity lost. End of the story.
Denis,
Thank you for your suggestion but in our case overexpression of proteasome subunit just to validate secondary experiment in paper is in my opinion wasting a money and really out of our filed since we are peptide chemists. Anyway I will try the best.
I didn't fully read this paper, but I got the impression they claim that b5 supports expression of catalyticaly active b1 and b2, don't they?
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2503187/
If detergents do not work (did you check that it is intact indeed?), how about some salts or maybe 2M urea? Are they covalently bound (disulfide bonds)?
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