Hi Hua,
Does your complex elute as a single peak from a gel filtration column or show a
monomodal dynamic light scattering profile? If not, it may be worth looking
into the method of complex formation. You can co-express the subunits, or
purify each subunits separately, and then mix them at a p
Dear CCP4 community members,
I've been trying to crystallize a protein complex that's very sensitive to
ionic strength, i.e., lower salt (~0.3M) will cause precipitation of the
complex but higher salt (~0.5 M) breaks the complex apart. The interaction
that holds the complex is probably mainly ion
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 y
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! ;)
Cheer
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.
> 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
Ooop
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:
http://www.cell.com/molecular-ce
