Here’s a summary of options people have sent me and some other related info I found on my own (email traffic has slowed down, so assuming all votes are in):
-Inteins: cut themselves off/out in presence of DTT or similar. NEB sells kits. -Sortase-His6: like inteins, but activated by Ca++ or triglycine. Hongyuan Mao, A self-cleavable sortase fusion for one-step purification of free recombinant proteins, Protein Expression and Purification, Volume 37, Issue 1, September 2004, Pages 253-263, ISSN 1046-5928, http://dx.doi.org/10.1016/j.pep.2004.06.013. (http://www.sciencedirect.com/science/article/pii/S1046592804002013) -CPD: cleaves itself off leaving 1-4 (even 0?) residues, activated by 5-20µM Inositohexaphosphate. Just like sortase, but activated with IP6 instead of Ca++/GGG. Possibly a problem in any organism which uses IP6 (if you know differently, please let me know.) 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 -FactorXA: when on N-term, no overhang—cleaves after IEGR. Expensive. -3C, aka prescission: very active at 4degC, better buffer range -New SUMO-protease-related proteases, seem to be dramatically more efficient than previous versions, also help with solublization: Frey, S.; Görlich, D.: A new set of highly efficient, tag-cleaving proteases for purifying recombinant proteins. Journal of Chromatography A 1337 95-105 (2014) Also, two nice sites I found listing some interesting options: http://web.expasy.org/peptide_cutter/peptidecutter_enzymes.html http://en.wikipedia.org/wiki/Protein_tag ******************************************* Jacob Pearson Keller, PhD Looger Lab/HHMI Janelia Farms Research Campus 19700 Helix Dr, Ashburn, VA 20147 email: [email protected] *******************************************
