Yongfu,
Small peptide tags usually don't interfere with protein folding and linker is not required. With removable tags, the protease site also serves as a linker. In most cases, putting a protease cleavage site such as TEV or PreScission site is good enough. The proteases (HRV3C and TurboTEV) are now available at a cost less than using thrombin (http://www.accelagen.com/proteins.htm#protease). So you can remove the tag and the proteases easily. For detection with Western blot, Flag, HA, Myc-tags are good and His-tag may be problematic since not all anti-His antibodies work well. However, His-tag gives an option to do binding in denatured condition. So for purification purpose, His-tag is probably your best bet. If you know that the precise N- or C-terminus is critical for protein function, put the tag on the other end or use Factor Xa cleavage site to remove N-terminal tag. You'll be surprised to find out that many proteins can be easily purified without using any tag. When we did the purification of SARS 3C protease, the purification scheme without using tag was actually simpler with higher yield. We have purified proteins for crystallography without using affinity tag from insect cells at an expression level of ~1 mg/L. From E. coli, a 3-step purification is generally sufficient to purify protein without using affinity tag. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 <mailto:[EMAIL PROTECTED]> [EMAIL PROTECTED] www.accelagen.com <http://www.accelagen.com/> _____ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Y. -F. Li Sent: Thursday, February 07, 2008 1:29 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tag, you're in! Flag, V5, etc Hello, I'd appreciate it if anyone could provide information (experiences or publications) on the following: 1. Put a tag such as FLAG, V5, etc etc, at the N- and/or C-termini, in order for specific detection, but not interfering with protein folding/structure; 2. Is a linker between the tag and target protein needed? What linkers (length, specific sequences) would you suggest? 3. What are the choices of such tags and why would you recommend them? Thank you for your time and sharing in advance. Yongfu Li
