Re: [ccp4bb] Tag, you're in! Flag, V5, etc
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
Re: [ccp4bb] Tag, you're in! Flag, V5, etc
Hi, I would like to point out, for the sake of fairness, that thrombin (high quality bovine thrombin such as sold by HTI for example) is still much cheaper *to use* than commercial TEV. One milligram of TEV, TVMV, AcTEV, and so forth can be used to cleave anywhere in between 10 to 100 mg of 'reasonably sized*' fusion protein, with typical ratio of around 1:25**. Thrombin is considerably more processive - 1 microgram of thrombin typically cleaves about 1 mg of fusion protein (again, reasonably sized) which is a ratio of 1:1000. The differences in processivity can be explained in terms of enzyme biochemistry and structure. These days everyone uses David Waugh's stabilizing mutation(s) of the TEV protease which in its native form deactivates itself in a few hours by means of site-specific autoproteolysis. TVMV protease does not undergo this process. Of course, TEV and other potyviral proteases offer unique advantages such as resistance to most protease inhibitors (yes, even E-64), extreme fidelity, and so forth. But the cheapest way to get them is to make them in-house (it's a 2-step procedure yileding about 100-150 mg of TEV or TVMV protease per liter of E. coli). Artem * all other things equal, in molar terms, the efficiency is the same for large or small constructs; but in weight terms larger fusions may be cleaved with higher efficiency. On the other hand, in practical terms large fusions can be tough to cleave due to the cleavage site steric protection by the sheer bulk of the construct. ** GST-TEV or MBP-TEV proteases can be even less efficient due to steric hindrance _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Chun Luo Sent: Friday, February 08, 2008 12:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Tag, you're in! Flag, V5, etc 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
[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
Re: [ccp4bb] Tag, you're in! Flag, V5, etc
If circumstances require a C-terminal tag, the intein system from New England Biolabs has worked very well for us. The pTYB1 plasmid encodes a fusion between your protein of interest, a viral intein (think protein intron) and a chitin binding domain. The fusion adsorbs to a chitin resin and all other proteins are washed away. The intein is engineered to not catalyze protein ligation but rather to release the protein in a thiol-directed manner. To cleave your protein from the fusion, simply add DTT, plug the column, and incubate overnight. The next day, push your unbound protein off the column: native protein with no tag residues left! The CBD-intein remain bound to the resin You don't get a lot of protein (5-10mg is more common than 50mg) and it is diluted to the volume of your column. On the other hand, it is pure enough to go right into trials upon concentration. At least that's been our experience. Andy (No affiliation with NEB) On 2/7/08 4:33 PM, James Whisstock [EMAIL PROTECTED] wrote: Hiya We usually add all N-terminal tags with a TeV cleavable linker. C-terminal tags always seem a pain to remove cleanly, because most highly specific recognition sequences (such as TeV) take advantage of P rather than P' specificity so you are usually left with a five (or so) residue tail. Actually has anyone any neat tricks for C-terminal tag removal? J -- Andrew M. Gulick, Ph.D. --- (716) 898-8619 Hauptman-Woodward Institute 700 Ellicott St Buffalo, NY 14203 --- Senior Research Scientist Hauptman-Woodward Institute Assistant Professor Dept. of Structural Biology, SUNY at Buffalo http://www.hwi.buffalo.edu/Faculty/Gulick/Gulick.html http://labs.hwi.buffalo.edu/gulick
Re: [ccp4bb] Tag, you're in! Flag, V5, etc
At least that's been our experience. Andy (No affiliation with NEB) Hi, everyone: Just to give both sides to this story, though, we've had phenomenally bad luck with the NEB Intein system (tagged on either the C- or N- terminus). The protein expressed and purified beautifully, and post-cleavage was pretty much a single band on a silver-stained gel. However, it was intractably misfolded, while purification with other, more 'traditional' tags (His6 or - dare I say it - *untagged* purification!) yielded fully soluble, folded protein. A few other labs around MIT have had similar experiences. So, for any technique there will be circumstantial data affirming or refuting its utility; take any success or failure story with the required ug of NaCl. =) Good luck purifying, Dave
