Totally agree with Chun. We are using a His tagged S219V construct that's very similary to the one described in the Waugh paper. To my experience, agitation, 37C incubation, low salt buffer, (etc.?), should all be avoided when using TEV. When using relatively large amount of enzyme(0.1~0.2mg/mL final, in total about 1/50 -1/10 of the total substrate by mol), we can often cleave our ProteinA fusions to nearly 90% complete on IgG beads. We do it at 4C overnight, elute, check the cleavage by SDS gel, if not complete, we add enzyme, cut O/N again, elute, check on SDS again. Then that's the 90% cleavage I am refering to. I also did O/N cleavage on column at 22C, and the cleave was quite good. In solution, my experience was positive too. In some cases even added at less than 1:200 molar ratio, the enzyme can still achieve a nearly complete cleavage at 4C in one or two days.
One thing that could greatly affect the cleavage is the accessibility of the cleavage site. Looking from the crystal structure of TEV protease, the Q in the ENLYFQ-G/S has better be at least 2-3 aa away from the folded protein domain coming up. I had one construct made by LIC, which had only one G outside the folded domain (revealed by later crystal structure), then it could not be cleaved at all. When two more amino acids were added, it cleaved fine. Zhijie From: Chun Luo Sent: Thursday, March 31, 2011 4:42 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Properly made TEV protease should work with or without rocking. We have done QC of our TurboTEV, a dual GST- and His-tagged TEV, under many conditions and used on hundreds of proteins. The most common cause of incomplete digestion is the insolubility of target protein or bad construct design. Shaking or agitation may oxidize the protein. TEV is a Cys protease that can be oxidized as well. Adding reducing agents or EDTA may well prevent some of the effects of agitation. Unlike plasmid DNA, protein solutions should be mixed GENTLY. Regarding the original question, 50% loss may not be a bad thing if it is about total protein. In our experience, it is not uncommn to have only 50% recovery after tag removal of a Ni pool. Some proteins aggregate after tag removal even it's His-tag. Again the loss can be a good thing. Chun Accelagen -------------------------------------------------------------------------------- From: Laurie Betts <laurie.betts0...@gmail.com> Sender: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> Date: Thu, 31 Mar 2011 11:11:33 -0400 To: <CCP4BB@JISCMAIL.AC.UK> ReplyTo: Laurie Betts <laurie.betts0...@gmail.com> Subject: Re: [ccp4bb] Question about TEV cleavage We have the same experience with the DO NOT AGITATE. We purify our own His-tagged TEV protease, and flash-freeze it IMMEDIATELY after IMAC prep at about 1 mg/mL. Laurie Betts UNC On Thu, Mar 31, 2011 at 10:37 AM, Charles Allerston <charles.allers...@sgc.ox.ac.uk> wrote: I echo this. Some time ago I was working on a target which seemed to precipitate when cleaving overnight with TEV. I wasted a fair bit of time trying to optimise cleavage conditions with a myriad of buffers. In the end, just by not agitating my solution, there was no precipitation and recovery was extremely good. I never agitate my solutions when cleaving now and have not had any problems with cleavage (to do with this issue, anyway) or recovery. cheers charlie Dr. Charles Allerston Genome Integrity Group Structural Genomics Consortium Nuffield Department of Medicine Old Road Campus University of Oxford OX3 7DQ http://www.sgc.ox.ac.uk/ -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Xiaopeng Hu Sent: 31 March 2011 15:23 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Question about TEV cleavage Our experience is do not shake the tube during TEV cleavage,I dont know why, but it does help. xiaopeng
