I have a fusion protein separated by thrombin cleavage site with GST. I eluted my fusion protein with 20mM Glutathione(reduced),50mM Tris(pH8),500mM NaCl,10mM CaCl2,10% glycerol and 5mM 2-ME. This complex buffer is actually suggested in the following paper: Kitamura et al., nsmb,2012. When eluted with this, thrombin in phosphate buffer (as suggested by the manufacturer) is added for digestion.
I face a problem of aggregation. Sometimes when thrombin is added, I can see a considerable amount of precipitation. Sometimes it may look ok (I carry out with more volume 3-5ml), but during the size exclusion the soluble protein is actually aggregated enough to be eluted at void volume. I also did on-column cleavage after binding in a GSH column, but the same result was observed after running SEC. Perhaps CaCl2 and 2-ME are needed for the fusion protein to attain proper solubility, but something wrong happens once it is cleaved!
Does anyone have any suggestions for this problem? Should I conduct dialysis after elution? Or Ion Exchange Chromatography after digestion? Any advice?
Hi Ananda,
The calcium is recommended for thrombin cleavage.
It seems likely that your protein is aggregating due to a loss of the GST tag (in which case your thrombin conditions are fine), and this is a quite common observation. Try adding any co-factor, ligand or substrate to your protein before cleavage, or screen other buffer conditions that do not promote aggregation of your protein.
Good luck, Christina
You can run the thrombin over a desalting column equilibrated with 50 mM tris to get rid of the phosphate and then add it to your fusion protein.
Thanks for your help.
What do you think if I omit CaCl2? why it is so important for GST-fusion protein? (I checked some companies suggest to do thrombin cleavage with Tris,150mM NaCl and 2.5mM CalCl2)
Hi Ananda,
The calcium is recommended for thrombin cleavage.
It seems likely that your protein is aggregating due to a loss of the GST tag (in which case your thrombin conditions are fine), and this is a quite common observation. Try adding any co-factor, ligand or substrate to your protein before cleavage, or screen other buffer conditions that do not promote aggregation of your protein.
Good luck, Christina
I would agree. Check it isn't a calcium phosphate precipitate first.
You can do this just by mixing the buffers under the same conditions (no proteins). Are you sure the tag was cleaved - have you run gels? Is the protein being over-proteolysed?
No it is not over proteolysed, it is the same when I incubate for 3 hr to overnight(did pilot test as well!); thrombin activity was pretty good, it digested amlost 100% of fusion protein. I would check it by mixing the buffer without protein and PBS to check Calcium phosphate salt formation. It is the big obstacle I am facing now :(
thank you Rob!
I would dialyse the sample before adding thrombin in PBS. Because of high CaCl2 it forms precipitation with phosphate buffer. Or use thrombin in some other buffer that doesn't make precipitation with CaCl2
Ca++ is not an essential cofactor that is required for thrombin activity. Just desalt/dialyse the thrombin to get rid of the phosphate.
Hi Steingrimur,
You mean phosphate buffer is not suitable for cleavage or to supect phosphates are responsible for aggregation even in the absence of Calcium? because, I can directly do all my purification in the same phosphate buffer/ Tris buffer(can bring thrombin to Tris as well!)
Hi Ananda, I'm not sure I understand the first part of your question.
I was just clarifying that thrombin doesn't require Ca for its activity. If the phosphate buffer in the thrombin prep is the source of your problems, then you can exchange it for most other buffers without worrying about losing all thrombin activity.
I wouldn't recommend changing the buffers in your purification scheme. If you have something that works-stick with it.
Hi Ananda,
How are you brother? It seems to me that we both are facing same problems. I am also dealing with same kind of fusion tag + protein system. I am also doing it in PBS buffer with thrombin digestion. Just after cutting, my protein gets precipitated. i am thinking to go for in-dialysis digestion. Here, i will put my protein + elution buffer (which contains 250 mM imidazol) in dialysis bag and add thrombin in it and then will dialyze it against PBS buffer. As the imidazol will decrease in the bag, thrombin will get activated slowly and will cut my protein from tag. Once i will get success, i will let you know but by the time i would suggest you to develop similar kind of experiment for you system. You can also try this strategy. Many of times it works.
Yogendra
Hi Ananda,
Thrombin from which company are you using? One from Novagen works best for me, which you can remove after digestion. Also some nonionic sugar detergent you can try to keep your protein in solution from aggregation in your buffer system where still thrombin can work.. Like CHAPS, MDM if it works for your protein. These detergents are very easy to dialyse when you may require to free from them and good for keeping activity intact mostly. I don't know if it will work for you or not. Have a good luck!
Nikhil Basu
Hi Jarosław, thrombin does not need Ca++ for its proteinase activity.
Generation of thrombin from prothrombin in vivo, however, is EDTA sensitive but it has nothing to do with proteinase activity.
I am not sure about precipitation of the fusion protein, but Phosphate buffer may form precipitate with CaCl2:- Ca3(PO4)2. It possible to check precipitation by mixing solution/buffer without proteins. When use CaCl2 its better do not use phosphate buffer, or use very low Cacl2 conc
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