How to remove thrombin from a digest

[BabcockHall]

Good Afternoon,

 

We would like to remove thrombin from a post-nickel column digest. However, our protein and thrombin have similar molecular weights; therefore, I don't hold out much hope for gel filtration. We think that cation exchange may work, inasmuch as our protein has a low pI. We are interested in co-crystallization experiments with a second protein.

 

There is a benzamidine column, but it is a little pricey. There is also biotin-labeled thrombin, but it has the same drawback. There is also APMSF as an alternative to PMSF, and this would at least inactivate the thrombin. Thanks for any suggestions.

 

[CharonY]

If immobilized thrombin or affinity columns are too pricey I guess you are limited to whatever separation method you have at hand (as new columns are not cheap either). Without knowing your target protein it is tricky to say but at least in theory reversed phase or ion exchange prep HPLC should do the trick. Even if inactive I would try to avoid additional contaminants in crystallization experiments as much as possible. Also, I typically check purity of overexpression purificaitions via LCMS as sometimes stuff stick to the column and get co-purified, which can be a problem if the desired proteins is not very highly expressed,

[BabcockHall]

Thanks. We were planning to do classical (low pressure) cation exchange, owing to a large difference in theoretical pI values between the two proteins, thrombin and the N-terminal and middle domains from FliM, which was well expressed. I imagine if this fails, we will go with a column of benzamidine or possibly biotin-labeled thrombin.

 

We are now routinely checking proteins with mass spectrometry. This is our first attempt at preparing this construct; therefore, we do not yet have HPLC conditions worked out.

[CharonY]

There is also immobilized thrombin that is covalently bound which saves a step over most biotin-thrombin protocols.

[BabcockHall]

Good idea. A long time ago, I immobilized alkaline phosphatase myself, but I doubt that it would be worth my time to learn how to immobilize thrombin. Better to go with the commercial product. Any thoughts about which is better, removal of thrombin via a benzamidine column versus immobilized thrombin?

[CharonY]

Typically with immobilized thrombin you will have less or no residual thrombin. In some cases digests seem to be less efficient (though not reproducibly so).