Hello,
I have a construct where a fusion protein is linked to my His-tagged protein of interest by the following sequence: ENLYFQ/D
I need to purify my protein of interest using a Ni-NTA affinity column. I tried to cleave out the fusion protein using a GST-Tev or an MBP-Tev, but none was efficient. All my tests are performed in 0.1% DDM (it is an intramembrane protein).
The Tev cleavage was performed in Tris-HCl 50mM, pH 7.8 with 150mM NaCl and 0.1% DDM.
I think that the tags in the Tev construct are big and are interfering with getting the enzyme close to the Tev recognition site.
I know that ENLYFQ/D is not the universal Tev cleavage site, but I have to have my protein starting with a D.
Do you know if there is any other Tev construct with a smaller tag, or no tag at all, or a construct that contains another cleavage site, like a thrombin site?
Thank you for your help.
Hi there,
The fact that the fusion protein is a membrane protein might be an issue for the efficiency of the digest by TEV protease (accessibility to TEV cleavage site might be hampered by the GST (or MBP) and the micelle in which membrane protein lies. Have you checked that the TEV is fully active on the similar protein (same cleavage site) issued from a fully soluble protein?
The most common plasmid is a a MBP fusion that has a TEV site, so it cleaves itself and you actually produce tag free TEV. I am sure you find the plasmid on Addgene.
The P1' site is not too bad for TEV protease as many side chains can be accommodated; Km/Kcat comparisons which was also done for caspase 3. Does this version have the mutation eliminating auto-inactivation? There should be some reducing agent and EDTA in the reaction since this is a cysteine protease. And yes there are relatively small molecular weight tags in the range of ~6- ~15 residues such as Strep II, T7, FLAG, S, HA, Myc, etc. but firstly be sure your current protease is actually working under optimal conditions so you know what course of action to take. If you're considering cost, purification, and yield then Strep II may be the best compromise. Are you been following any literature from others who have done similar cleavages for membrane proteins?
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