- This topic has 8 replies, 8 voices, and was last updated 10 months, 1 week ago by Frederico Silva.
January 27, 2023 at 12:56 #4601
By routine we remove our tags with 3C and TEV but have now a case where we have a thrombin cleavage site.
Does anybody has experience with thrombin cleavage and Removal?
I was wondering about what the best way of going about it for instance in terms of what thrombin to use, what cleavage conditions (as to maximize activity but also specificity) and about the best way to purify after cleavage, etc
Thank you in advance,
January 27, 2023 at 13:58 #4608
We have been working with thrombin cleavage for some proteins and it works well although the protease is quite expensive.
We were using Thrombin from GE and molar ratios of 1:100 were cleaving 100%.
Thrombin is sensitive to high concentrations of salt and presence of Imidazole so I highly recommend dialysis if the protein is eluted from IMAC.
After cleavage we were using flowthrough IMAC as thrombin is His-tagged.
hope it helps.
January 27, 2023 at 14:23 #4609
I try to spread the word that people should switch their cleavage sites with proteases that are tagged and homemade, but it is difficult if people always go back to the commercial vectors (however much easier now since people can just synthesize cassettes).
For the removal, if necessary, I typically just do a SEC if the size permits. For conditions, Ive cleaved in pretty much every condition PBS, HBS, TRIS-NaCl. The amount, is like all proteases dependent on substrate. But typically 1:100-500 is the range I am at.
January 27, 2023 at 14:25 #4610
I used thrombin cleavage on only one GST-tagged protein. We bought thrombin as a powder (probably also from GE) and reconstituted it to 1000 u/ml in 1x PBS.
We performed the cleavage at room temperature with 10 U/mg protein. This gave 100% cleavage.
The thrombin was removed during the following purification steps. We did not actively remove it.
January 27, 2023 at 16:02 #4612
sandrine vadon le goffParticipant
We used to do this on immobilized thrombin on agarose (Thrombin CleanCleave kit, Sigma), which is quite convenient to separate products from thrombin
1 mL is more than enough for 10 mg of protein. Add CaCl2 2 mM and incubate overnight at 4°C, + 1 hour rt. The cleavage products are recovered from the column by gravity (+wash)
In these conditions we had 100% cleavage
January 27, 2023 at 16:03 #4613
Regarding thrombin removal after the cleavage, you could pass your protein solution over some benzamidine Sepharose which will bind thrombin.
January 27, 2023 at 17:06 #4617
We have used the restriction grade thrombin from Merck/Millipore in several projects and it worked really well. They claim 1 unit of protease is required per mg of fusion protein, but we have used it more diluted than that and still got 100% cleavage after overnight incubation at 4*C. For the removal we also just do a SEC as Kelvin suggested (if the size differences allow that). If not, benzamidine sepharose is our preferred option as well.
January 27, 2023 at 18:45 #4621
Another protocol just so you have a lot of choices!
we use Thrombin T4648-10KU from Sigma,
Diluted to be aliquoted at 1U/µl (100µl aliquots) and frozen -70°c
Then use 10U to cleave 1mg, directly on the column where is bound your His or GST protein, without any flow, overnight at room temperature.
Thrombin is then removed later during purification steps or using benzamidin column
Have a good week end
January 31, 2023 at 13:01 #4624
Thank you so much for your prompt and kind help.
You (we) rock!
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