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
