Hello,
I am trying to cleavage my protein from GST-tag by using Thrombin with its site ( L-V-P-R-G-S) which is provided by plasmid. However, I am confused because I saw after running my protein sequence in ExPASY tool that my protein has also a cleavage site for thrombin (arg-gly). So is the R-G amino acids seq enough to cleavage by thrombin?
Coen has already answered most of your question, but a fantastic site for evaluating the specificity of different proteases is the MEROPS database. I've linked the entries for thrombin and for TEV protease.
Is there a structure available for your protein? Remember that for a cleavage to occur, the sequence has to not only match the consensus, but needs to be accessible to the protease. If your R-G is in the middle of a beta sheet or alpha helix, it's very unlikely to cleave. If it's in a flexible loop or random coil, you're in trouble.
http://merops.sanger.ac.uk/cgi-bin/pepsum?id=S01.217
http://merops.sanger.ac.uk/cgi-bin/pepsum?id=C04.004
Hi there,
The occurence of thrombin cleavage at secondary site (like the RG you have) is dependent on the amount of thrombin used. I would suggest you to try lower thrombin concentration on your protein and see what happens. Usually, 1U is enough to efficiently cleave 1mg of target protein (overnight at 20°C). If that's the condition you have used, I would go for less thrombin.
I hope it helps.
Dear Ahmed,
Unfortunately, the activity of thrombin is definitely not limited to one single site. You can already tell by the wide variety of substrates it can cleave: fibrinogen, PARs, cofactors FV and FVIII, Factor XI, and those are only example targets without thrombomodulin involvement. The R is the final amino acid of a consensus sequence that thrombin targets. Many serine proteases (kallikreins, plasmin) behave in a similar manner. However, not all in silica predicted cleavage sites are actually cleaved in real life. There is only one way to find out: produce, incubate with thrombin and see whether your target protein is cleaved. If you're still in the 'construct designing' stage, you may want to introduce a TEV protease cleavage site (ENLYFQS) between protein and tag as an alternative. This sequence is much more stringently recognised by its target protease and differs greatly from that of the serine proteases.
Good luck,
Coen
PS I included a great FAQ on TEV protease
http://mcl1.ncifcrf.gov/waugh_tech/faq/tev.pdf
Coen has already answered most of your question, but a fantastic site for evaluating the specificity of different proteases is the MEROPS database. I've linked the entries for thrombin and for TEV protease.
Is there a structure available for your protein? Remember that for a cleavage to occur, the sequence has to not only match the consensus, but needs to be accessible to the protease. If your R-G is in the middle of a beta sheet or alpha helix, it's very unlikely to cleave. If it's in a flexible loop or random coil, you're in trouble.
http://merops.sanger.ac.uk/cgi-bin/pepsum?id=S01.217
http://merops.sanger.ac.uk/cgi-bin/pepsum?id=C04.004
What I have done in the past where the tag has been an issue is to restrict out the gene from the vector and ligate it into a vector with a much smaller affinity tag, like Novagen's His6-tag or S-tag. If I recall correctly, I dropped my gene out of a pGEX vector and put it into pET21 (or pET28 or pET29), and it ended up being in-frame. With the GST tag cleaved off, you would end up extra amino acids still appended to your recombinant protein so leaving a small His6 or Stag (or Flag-tag for that matter) might not be too bad.
Thank you for all. Mr. Croll I think I am in trouble because R-G is at the first 14 amino acids. Is there any suggestions?
Do you have access to mass spectrometry? If so, you could run a trial cleavage and then see if your masses come out as expected. Don't forget to also run some on a reducing gel to check for internal cleavage sites.
If it does turn out to cleave your N-terminal site, the next question to ask yourself is, is that a problem? Are the first 14 residues critical to the function of your protein?
Unfortunately No. the first 14 a.a are important to my experiment. I am checking protein- protein interactions. Thanks though
@Tristan Croll: What is the reason behind saying that "If it's in a flexible loop or random coil, you're in trouble"?
II is a serine protease which plays a central role in the conversion of fibrinogen to fibrin in the clotting cascade. Substrate sensitivity t o Arg-X in fibrinogen appears to require a proline bend with Leu-Val-Pro on one side of the bend and Arg-Gly- Ser on the other..
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