Dear all, I agree with all said so far: Here are some points from my experience and discussion on this topic.
Most of the lab's due use TEV or 3C, depending on the cleavage site in their constructs and make their purification strategy work with what they have. Here are some points from my side: * 3C protease is much faster in cleaving the fusion protein in the 1:100 mass ratio (a test we use in cleavage protocols, even though we should use a molar ratio. Consider the difference between 20 kDa protein and 200 kDa protein, it is 10x in the amount of substrate difference) 3C cleaves in 30 mins more than 95% of the protein at 4-8C. TEV does need much longer, and as default, people go for overnight dialysis and cleavage simultaneously. Sometimes that can be too long for sensitive proteins and lead to precipitation due to chance in the buffer salt concentration etc, for an extended period. Quite prominently is observed for Nucleic acid binding protein, due to lowering the salt for IEX column and precipitation surprise overnight. * Neither 3C nor TEV requires a reducing agent, which is essential to know as many people are working with S-S-containing proteins, and they take an aliquot of the protease purified in the lab without knowing there was a reducing agent in the storage buffer. Then surprises can be observed. Consider the concentration of 1 mM DTT; even 10 or 100 diluted is 10 µM at least. * Same with a chelating agent like EDTA, no need to be added to the storage buffer; the danger comes when you cleave ion-containing proteins like Zn-fingers. * 3C is much more efficient in on/column cleavage (as was already mentioned). This simple trick gives much better purity than Imidazole elution, especially when the Ni-column is not saturated and many contaminants are bound. Releasing the protein with the protease makes it much cleaner. Most of the protease has a His-tag, which allows at the same time to bind it to do beads (of course this slows down a bit the cleavage therefore mixing is required). Alternatively, GST-(only)-tag protease is better used for the cleavage on Ni beads and then captured on the GSH resin. * 3C protease is much more efficient for cleaving in the presence of detergent, there is an extensive study on that topic (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/) * Both proteases are easy to produce, as mentioned, and easily 100-300 mg can be obtained from 1 L culture (TB or auto-induction with OD= 8-16) * 3C protease is not happy at high protein concentrations (>4 mg/ml) in low salt buffer (150 mM). Therefore huge loss is observed if, during the Ni-elution, low salt buffer is used as the protein comes from the Ni-NTA at a concentration 15-20 mg/ml. Please use at least 500 mM NaCl for that step or better for any step from lysis to elution. * And yes both proteases have cross-activity and can cleave the opposite target site with lower activity. These are the few points coming from the top of my head. I hope they are helpful and wish everyone happy cleaving of the fusion proteins :) Kind regards, Nikolay Nikolay Dobrev Postdoctoral Fellow @ Wilmanns group EMBL Hamburg, c/o DESY, Building 25A, Notkestraße 85, 22607 Hamburg, Germany T +49 40 89902 165 | M +49 173 684 0532 twitter.com/emblevents https://twitter.com/emblevents |http://facebook.com/embl.org | http://youtube.com/user/emblmedia Visit http://www.embl.org/events for a complete list of all EMBL events. > On 12/07/2022 11:51 PM Lior Almagor <lior.alma...@gmail.com> wrote: > > > In my experience, 3C can partially cut TEV sites as well. If using > sequentially, it is best to plan to use the TEV first. > > > > > On Dec 7, 2022, at 2:42 PM, Lau Kelvin > <00005aaf8435dbef-dmarc-requ...@jiscmail.ac.uk > mailto:00005aaf8435dbef-dmarc-requ...@jiscmail.ac.uk > wrote: > > I agree. I think it depends on the lab and vectors and personal > > preference. > > > > But David, have you used them sequentially? I have once tried to > > make a His-GST-ENLYFQ-3C construct, and I found that it was self cleaving > > during expression. However I have never tried to replicate the results in > > vitro. > > > > > > > > -- > > Kelvin Lau > > Protein production and structure core facility - PTPSP > > EPFL SV PTECH PTPSP > > AI 2146 (Bâtiment AI) > > Station 19 > > CH-1015 Lausanne > > Switzerland > > Email: kelvin....@epfl.ch mailto:kelvin....@epfl.ch > > Phone: +41 21 69 34494 > > > > > > > > > On 7 Dec 2022, at 21:38, David Briggs > > <david.bri...@crick.ac.uk mailto:david.bri...@crick.ac.uk > wrote: > > > Hi Gloria, > > > > > > Both can be made very easily in E.coli. > > > Both are active at 4°C, but especially 3C, I think. > > > > > > I have plasmids for both somewhere in the freezer (you might > > > find someone closer to you who can send HRV3C, but if you cannot, let me > > > know off list). > > > > > > I don't see any particular benefit of one over the other, but > > > having both in your freezer means you can cleave off tags sequentially as > > > needed by your purification strategies. > > > > > > HTH, > > > > > > Dave > > > > > > Dr David C. Briggs CSci MRSB > > > Principal Laboratory Research Scientist > > > Signalling and Structural Biology Lab > > > The Francis Crick Institute > > > London, UK > > > ==http://about.me/david_briggs > > > > > > > > > --------------------------------------------- > > > From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK > > > mailto:CCP4BB@JISCMAIL.AC.UK > on behalf of Gloria Borgstahl > > > <gborgst...@gmail.com mailto:gborgst...@gmail.com > > > > Sent: Wednesday, 7 December 2022, 20:26 > > > To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK > > > <CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK > > > > Subject: [ccp4bb] TEV vs HRV3C > > > > > > > > > External Sender: Use caution. > > > > > > Hello my fellow structural biologists, I am contemplating > > > why some choose the HRV3C protease site over TEV for their fusion > > > proteins. Does anyone know? Can HRV3C be made easily in homelab? Does > > > anyone have a plasmid? Thank you, G > > > > > > > > > --------------------------------------------- > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > > > > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > https://eur03.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=05%7C01%7C%7Cb865bd537cfa41af3b0f08dad8914aeb%7C4eed7807ebad415aa7a99170947f4eae%7C0%7C1%7C638060415788656699%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000%7C%7C%7C&sdata=M1H4lHtUxJ1WZR0%2B73eYsZCc8%2FBDO8pWcV0yjFgomHk%3D&reserved=0 > > > > > > > > > > > > The Francis Crick Institute Limited is a registered charity > > > in England and Wales no. 1140062 and a company registered in England and > > > Wales no. 06885462, with its registered office at 1 Midland Road London > > > NW1 1AT > > > > > > > > > > > > --------------------------------------------- > > > > > > To unsubscribe from the CCP4BB list, click the following link: > > > > > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > > > > > > > > > > > --------------------------------------------- > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > > > > > > --------------------------------------------- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
