Re: [ccp4bb] off-topic: tag removal
Hi Phil, Depending on the characteristics of the c-terminal region of interest, you might try a carboxypeptidase. These enzymes cleave residues from the c-terminus and stop at various motifs, depending on the specific enzyme. There are several available commercially, each having a slightly different activity. Also, the following paper may be of interest: Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins José Arnau, Conni Lauritzena, Gitte E. Petersena and John Pedersena Protein Expression and Purification, Volume 48, Issue 1, July 2006, Pages 1-13 Good Luck, Mike - Original Message - From: Philipp Ellinger filu...@gmx.de To: CCP4BB@JISCMAIL.AC.UK Sent: Wednesday, February 23, 2011 1:17:33 AM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] off-topic: tag removal Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] off-topic: tag removal
I haven't used it personally, but I've heard some people engineering a his SUMO tag on to the protein and then use SUMO protease to cleave it off. The protease apparently makes very clean cuts and no extra residues are left. Best of luck, Peter
Re: [ccp4bb] off-topic: tag removal
Based on Peter's suggestion: yes: the SUMO protease does an efficient job for removal of the tag. Lucigen (www.lucigen.com) does sell new generation of cloning vectors (IPTG and Rhamnose inducible) with SUMO tag as well as SUMO protease included in their kit. The novel Expressioneering Technology offers very easy cloning and protein expression tools. Thanks and Good Luck. -Saurabh On Thu, Feb 24, 2011 at 12:15 PM, Peter Hsu hsuu...@u.washington.eduwrote: I haven't used it personally, but I've heard some people engineering a his SUMO tag on to the protein and then use SUMO protease to cleave it off. The protease apparently makes very clean cuts and no extra residues are left. Best of luck, Peter --
[ccp4bb] off-topic: tag removal
Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] off-topic: tag removal
See the following reference- Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Philipp Ellinger [filu...@gmx.de] Sent: Wednesday, February 23, 2011 3:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] off-topic: tag removal Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] off-topic: tag removal
Do you have the crystal structure ? Then look for a surface loop/exposed area and insert the His6-tag there. Jürgen On Feb 23, 2011, at 4:17 AM, Philipp Ellinger wrote: Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] off-topic: tag removal
I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? C-terminal intein fusion is a self-cleavable tag that cuts cleanly. NEB sells kit for intein-chitin binding domain fusion for affinity purification but of course you can add any other tag of your liking. - Dima
Re: [ccp4bb] off-topic: tag removal
Hi Phil, Must you tag your protein? Have you tried to express it without a tag? Perhaps you can purify the un-tagged protein with a series of chromatographic (or other) steps - particularly if it expresses well. Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Wed, 23 Feb 2011, Philipp Ellinger wrote: Dear all, I have a question concerning removal of a his-tag sequence. We have crystallized a protein with an important feature at the C-terminal part of the protein. Unfortunately, we cannot express it with a N-terminal his-tag, only with a C-terminal his-tag. Therefore we are looking for a protease which cleaves off the sequence without leaving any extra amino acid on the C-terminus of our protein. Meaning we obtain really the wild type protein. Does anyone know about a protease or cleavage site which is completely removed? Many thanks in advance Phil
Re: [ccp4bb] off-topic: tag removal
Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009 Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119 Unless the protein in question happen to be Leu as a C-terminal residue, this tag won't give wild type C-terminus after cleavage. - Dima
