Incomplete translation of N-term His-tagged protein in E.coli
(by ben.long from yourfinger.anu.edu.au)
Wed Nov 7 22:05:15 EST 2007
I am expressing a protein in E. coli which has a cleavable N-terminal
His-tag. The purified IMAC elaute prior to tag cleavage contains the
tagged protein plus a large number of lower mass bands on SDS-PAGE.
These masses all drop accordingly in size when the tag is cleaved,
suggesting they are incomplete translation products (i.e. N-terminus is
intact but C-terminus is shortened) of the protein I'm after. Can anyone
offer any suggestion about, say, growth conditions or other other
treatments which might help avoid this? I am growing cultures overnight
at 23 °C with 0.5 mM IPTG induction and harvesting using pretty standard
methods (spin, lyse in IMAC binding buffer, etc). The cleavage requires
a specific protease, so a protease cocktail is not used, just PMSF.
Nonetheless, I am going to try using a cocktail next time to see if this
might be a C-terminal protease problem. I am also expressing a short
form of the same protein (which has a portion of the N-terminus missing)
which does not appear to have the same problem, but this may be due to
relatively low yields resulting from an apparent dimerisation and
inaccessibility to IMAC in the short form. Ideas would be welcome.
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