expression of a fusionprotein
cbookste at nospammedicine.bsd.uchicago.edu
Mon Dec 15 09:21:02 EST 1997
Andrea, We have made many different fusion proteins and the construction is
simple compared to successful expression. Some were absolutely perfect by
sequence and we never found a way to either express or purify.
Some methods which have worked when the most straightforward expression
yielded no fusion protein:
Test several clones from the original ligation looking at whole cell
lysate of induced versus uninduced. For reasons unknown, some clones
perform better than others.
Carefully test several different temperatures of incubation of the bugs
Or test induction at 30 rather than 37 degrees.
The amount of IPTG may also be crucial.
Try omp, Deg minus bacteria.
Induction time can be crucial...too long may contribute to proteolytic
breakdown of the recombinant protein.
Try induction only with freshly transformed bugs.
Over sonication can be more detrimental than too little.
You may be losing the recombinant protein in the binding step. The
fusion may have altered the core protein folding so that it does not bind
efficiently. Check all washes, bound and eluted fractions. (If this is
the case, either gel purify or try another fusion.)
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