Hi Giulliana,
You can check our general protocol for TEV digestion (http://www.accelagen.com/TurboTEV-protocol.htm). Once you mixed TEV with protein solution, let it sit for digestion. No shaking or rocking. We found shaking or rocking sometimes reduce the efficiency. Some constructs just refuse to be cut by TEV. You may run analytical SEC or light scattering to see if your protein forms dimer or oligomers. The digestion is less or none for aggregated constructs. Find a buffer that your protein is most happy with. Some detergents work well with crystallization. TEV itself should be very stable in majority, if not all, of the buffers used for recombinant protein production. At least we never seen any issues with TurboTEV. As Karsten mentioned, HRV3C protease is more robust than TEV for tag cleavage. However HRV3C cut leaves 2 extra residues. Cheers, Chun From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana Rangel Sent: Saturday, May 2, 2015 10:57 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel
