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Old 03-27-2011, 04:46 PM
Pipette Filler
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Hi there,

I've just cloned a couple of constructs using the pGEX system to purify protein for crystallisation trials. So it looks like I'll need tons of protease (TEV, prescission and thrombin) soon enough. However, one of my supervisor's grant
applications has just been rejected and we're a little short of money. I wonder
whether anyone could think of a straightforward way to obtain protease activity, ideally without having to go through multiple protein preps.

I've been told that there are ways to express proteins fused to outer membrane
components, for example the AIDA system of autotransporters, so couldn't i
just clone an AIDA-protease construct and lyse the cell after inducing expression, spin it down to remove in the supernatant cytosolic proteases
and use the membrane debris for my cleavage reactions? Any thoughts would
be greatly appreciated.
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