You can use a redox system which should maintain disulfide bonds, but still offer enough reduction to give TEV active. Use 3mM reduced glutathione/0.3mM oxidized glutathione. Add the protein to TEV so the DTT is rapidly diluted.
We started to make our own when it rapidly became used my nearly everyone else in the lab. Typically 6l of cells which produce enough TEV for a lab of 6-8 people for about 1-1.5 yr. I believe we obtained the plasmid from David Waugh (https://mcl1.ncifcrf.gov/waugh_tev.html) sometime last decade. Best of luck, Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 -----Original Message----- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nicola Evans Sent: Wednesday, February 13, 2019 1:46 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] TEV Protease in low reducing agent? I am interested in purifying proteins with 1 or 2 disulphide bonds, and have been using enterokinase to cleave off N-terminal tags but we have had issues with poor cleavage and want to try TEV cleavage instead. However TEV protease is usually kept in a high amount of DTT and I am concerned about reducing the disulphide bonds in my purified proteins. I have used TEV protease before with 0.25mM TCEP and it worked well. Is there a way to use TEV with very little or no reducing agent? Perhaps by optimising conditions such as adding glycerol? We were thinking of buying in commercial TEV protease so any advice on that is also welcomed (is there any merit, except cost, to making it ourselves?) ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
