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 <gborgst...@gmail.com> 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 pjl...@gmail.com pj...@drexel.edu ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
