Another option would be to use a protease immobilized in some resin (for instance GST-tagged TEV protease bound to GSH-Agarose resin, in column or in batch), so that no protease would be free to co-purify with your protein or peptide. HTH, Javier
On Thu, Sep 20, 2018 at 5:35 PM, Patrick Loll <[email protected]> wrote: > 1) Use a different protease, e.g. SUMO protease, which is sufficiently > specific that it’s unlikely to cause any problems even if a little bit is > carried along. See, for example (shameless plug #1): DOI: > 10.1016/j.pep.2006.12.006 > > 2) Use intein cleavage. NEB sells a vector that lets you fuse an intein & > a CBD at the C-term end of your POI; we’ve made a similar vector with a > His-tag (shameless plug #2): DOI: 10.1021/ja208755j > > 3) Just assure your colleague that you guys are good protein chemists, so > don’t worry, be happy. > > There are chemical cleavage methods (e.g. cyanogen bromide), but they > aren’t particularly gentle, so if your 110-residue peptide is meant to fold > into a native structure, I’d approach them with caution (on the other hand, > if the peptide ISN’T meant to be folded, then just purify via > reverse-phase, and you’ll for sure get rid of protease contaminants). > > Pat > > > > On 20 Sep 2018, at 4:17 PM, Gloria Borgstahl <[email protected]> > wrote: > > > > Hello, friends in crystallography, > > A colleague just asked me this question. He is worried about trace > > protease interfering with the receptors he is studying in cell-based > > experiments using a 110 amino acid protein we made for him. He has > > been unable to make the peptide synthetically. The company is having > > trouble getting that to happen. Any ideas? Happy Thursday, G > > > > ######################################################################## > > > > To unsubscribe from the CCP4BB list, click the following link: > > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > > ------------------------------------------------------------ > --------------------------- > Patrick J. Loll, Ph. D. > Professor of Biochemistry & Molecular Biology > Drexel University College of Medicine > Room 10-102 New College Building > 245 N. 15th St., Mailstop 497 > Philadelphia, PA 19102-1192 USA > > (215) 762-7706 > [email protected] > [email protected] > > ######################################################################## > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- Dr. Javier M. González Instituto de Bionanotecnología del NOA (INBIONATEC-CONICET) Universidad Nacional de Santiago del Estero (UNSE) RN9, Km 1125. Villa El Zanjón. (G4206XCP) Santiago del Estero. Argentina Tel: +54-(0385)-4238352 Email <[email protected]> LinkedIn <https://www.linkedin.com/in/javier-m-gonzalez-inbionatec> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
