Thrombin recognises the consensus sequence Leu-Val-Pro-Arg-Gly-Ser, cleaving the peptide bond between Arg and Gly. This is utilised in many vector systems which encode such a protease cleavage site allowing removal of an upstream domain. Predominantly the domain to be cleaved is a purification tag such as a 'His-Tag'

A problem with many commercial thrombin sources is secondary protease activity.
If this were due to thrombin alone then this would not, in general, be an
insurmountable problem, since this can relatively easily be dealt with (with
benzamidine and via a benzamidine sepharose column, vide infra). However human
thrombin is one of the most active site-specific proteases. Our experience (and those
of our colleagues in Toronto) suggests that such secondary activity arises from other
proteases present in the purchased thrombin. Al Edwards' group generally further purify purchased thrombin with a Mono-Q ion exchange chromatography step which is an advisable and simple
further step.

In any case, what follows is a general guide for cleavage. It will have to be amended
from one protein expression system to the next.

1) Use the highest grade human thrombin available from Calbiochem. - you'll need
somewhere between 1 and 10 units per mg of recombinant protein.

2) After the Ni-column purification it is advisable to exchange (e.g. dialyse) the protein
into a low-medium salt buffer (I use 20 mM Tris-HCl, 100mM NaCl all at pH 8,
no protease inhibitors!). High Imidazole (i.e the Ni-NTA column elution buffer) inhibited
the cleavage in my case.

Further guidelines for factors affecting thrombin activity and other information
such as the effect of denaturants can be found in Novagen's pET system manual. Also, Novagen's site
contains a wealth of information on all aspects of protein purification and is generally a good resource.

3) Add thrombin preferably at room temperature) and shake gently.

To monitor the reaction - (optional)
4) Monitor reaction the first time by taking aliquots (10 ul) at various time intervals and,
via SDS PAGE look for a gel shift - in my case the protein is 2 KDa lower in molecular weight when cleaved so is easily detectable (provided your protein is in the 0-40 KDa
range). Keep some uncleaved protein as a control and spot a mixture of uncleaved and putatively cleaved in one lane. You may need to allow the gel front to run into the gel buffer solution to get sufficient resolution.

Other assay methods include mass spectrometry - there is a mass difference  between
cleaved and uncleaved protein which should be easily detectable.
In any event the next step separates cleaved and uncleaved proteins so step 4 should not
be necessary beyond the first time. This step was added to allow you to gauge your
reaction timescale and not leave your protein needlessly sitting in a thrombin containing solution for an excessive period.

6) Separate the cleaved protein from any uncleaved protein and the his containing
peptide by running the whole mixture through another Ni-NTA column under the same
conditions as the first time. The cleaved protein should just flow through and
uncleaved protein (which you may wish to recover) will bind to the column and can be
eluted with imidazole.

7) Run the collected fraction through a pre-equilibrated (in buffer of choice and with 20 column vol. of equilibration) Benzamidine Sepharose column (1ml of slurry, purchase from Pharmacia). This should bind residual thrombin preventing secondary activity from this source and some others. Collect the flow through.

8) Add PMSF (1mM, I suggest using a freshly prepared 100 mM ethanolic stock solution)
and benzamidine (0.1-1mg/ml from a 50 mg/ml aqueous solution)
and any other protease inhibitors you deem necessary.

9) Monitor process by SDS-PAGE.
 
 

 Last Update: John Christodoulou, May 3 2000