Le 26/02/2011 03:51, Dima Klenchin a écrit :
It surely is not. An N-end rule has to do with ubiquitination, and it is
absent in E.coli.
Not true. There is indeed and N-end rule in prokaryotes, including E.
coli. Mediated by the ClpP protease-based system. See:
It surely is not. An N-end rule has to do with ubiquitination, and it is
absent in E.coli.
Not true. There is indeed and N-end rule in prokaryotes, including E.
coli. Mediated by the ClpP protease-based system. See:
http://www.cell.com/molecular-cell/abstract/S1097-2765%2808%2900692-8
If you want a ClpP minus strain you can get it from the Keio strains.
http://cgsc.biology.yale.edu/index.php
http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp
For expression with the T7 promotor, you will need to use the λDE3
Lysogenization Kit or use a Arabinose induction plasmid instead.
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! ;)
Factor Xa will work depending on the exact sequence. If your sequence starts
MRS... and the start methionine is important and not removed then yes it will
work when you clone it into a Factor Xa site. If however the start sequence
starts RS... and the start methionine is actually removed, when
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,
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
