You could try adding TEV to your protein just prior to crystallisation. I produced 2 equivalent structures this way; one with the tag clearly visible packing between molecules in an I centred space group and a 2nd (with TEV added) in P21 where the tag was no longer visible (the packing suggested it would not be accommodated and therefore cleaved). I don’t think many people try detagging their protein in conditions as extreme as those in a crystallisation trial but in the end that’s where the protein needs to be happy. Yes, there are lots of caveats with this technique, not least sub optimal cleavage conditions, but it’s a very simple experiment to do.
Dave From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana Rangel Sent: 02 May 2015 18:57 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 ________________________________ AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
