Incomplete translation of N-term His-tagged protein in E.coli

Bean Long via methods%40net.bio.net (by ben.long from yourfinger.anu.edu.au)
Wed Nov 7 22:05:15 EST 2007


Hi all,

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.

Cheers,
-- 
Bean

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