Dear Jerry, Whats about an N-terminal His-Tag with an Xa factor cleavage site behind it...
After IMAC you only get protein with the entire N-Terminus (His-Tag), afterward digest the protein with Xa factor...it won¹t leave any additional amino acids C-terminally! You¹ll get your protein! ;) Cheers, Christian Am 2/26/11 2:15 AM schrieb "Jerry McCully" unter <for-crystallizai...@hotmail.com>: > Dear ALL: > > Recently I am expressing one protein in BL21(DE3) and the protein > undergoes N-terminal degradation. > > I am trying to keep this crucial N-terminal tail on the protein, which > has MRS at the first 3 positions. > > Digging in to the literatures, I found the N-end rule, which tell that > the proteins bearing N-terminal Arg or lys have much shorter half life. > > I am not sure whether this is my problem. However, I want to found one E. > coli mutant strain that lacks aat and is unable to degrade N-end rule > substrates that bear N-terminal Arg or Lys. > > Does anybody know such strains? > > Thanks a lot and have a nice weekend, > > Jerry, >
