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Topic: Does 10 mM glutathione inactivate thrombin?  (Read 2215 times)

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Offline Babcock_Hall

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Does 10 mM glutathione inactivate thrombin?
« on: August 27, 2015, 12:47:46 PM »
We are making a GST-fusion protein for the first time.  Typically these are eluted from an affinity column with 10 mM glutathione:  From an article on the subject, "10 mM glutathione buffer: 50 mM Tris, 10 mM reduced glutathione, pH 8.0 (make fresh daily)" 
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584333/

For some applications the next step is removal of GST tag by cleavage with a protease (thrombin in our case).  Thrombin contains several disulfide bonds.  Some years ago, I looked into the issue of reducing agents and how they might inactivate thrombin.  My recollection is that low concentrations of certain biochemical reducing agents could be tolerated, but high concentrations could not be.  What I am having a hard time learning is whether or not 10 mM glutathione will reduce and inactivate thrombin.  On the one hand, 10 mM is a decent concentration; on the other hand, glutathione might be too bulky to react with thrombin quickly.  Ordinarlly I would just do a buffer exchange, but we are trying to minimize the number of steps of things like buffer exchanges, because our protein can drop out of solution under conditions that we have not been able to predict or control yet.

Offline Yggdrasil

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Re: Does 10 mM glutathione inactivate thrombin?
« Reply #1 on: August 27, 2015, 04:12:36 PM »
Have you considered eluting from the glutathione column via thrombin cleavage instead of performing the elution and cleavage as separate steps?

Offline Babcock_Hall

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Re: Does 10 mM glutathione inactivate thrombin?
« Reply #2 on: August 27, 2015, 07:19:26 PM »
Have you considered eluting from the glutathione column via thrombin cleavage instead of performing the elution and cleavage as separate steps?
Yes, if you mean perform an on-column cleavage.  One cannot easily monitor the cleavage reaction that way, and I have heard that yields can suffer.  However, it might not be a bad idea under the circumstances to move in this direction, but I was thinking of moving to on-column cleavage after working it out in solution.
« Last Edit: August 27, 2015, 10:01:10 PM by Babcock_Hall »

Offline Yggdrasil

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Re: Does 10 mM glutathione inactivate thrombin?
« Reply #3 on: August 28, 2015, 01:10:00 PM »
You could assess the cleavage efficiency by washing the cleaved protein from the column, then the eluting the remaining GST plus fusion protein with glutathione.  Running on a SDS-PAGE gel would tell you how much protein remained uncleaved.  Of course, this would not allow doing timepoints as in the case of an off-column reaction.

Offline Babcock_Hall

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Re: Does 10 mM glutathione inactivate thrombin?
« Reply #4 on: September 04, 2015, 11:27:03 AM »
We are considering buying a benzamidine column.  One question that arises is whether or not benzamidine would bind thrombin that has been inactivated by PMSF.  Another question is the best way to tell whether one's thrombin-removal technique is working.  Does anyone have experience with a good synthetic substrate, such as this one?  http://www.anaspec.com/products/product.asp?id=50223

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