Expression of a 300kDa protein possible?
pd1 at mole.bio.cam.ac.uk
Tue Apr 4 07:29:37 EST 2000
Christina Philipp wrote:
> Hello Ng,
> until now, I tried two methods to express my protein:
> 1. TNT T7 Quick Coupled Transcription/Translation System (Promega).
> The luciferase control worked well, but I did not see my desired
You don't say what you did get with the IVT: a smear of shorter products
nothing at all? (The latter would indicate a problem with the construct
rather than retic struggling to make such a large protein).
The largest protein I've made/seen made in retic is up around the 250
(albeit with quite a lot of shorter crap too), so 300 should be
There are a few things you can do to improve your chances:
1) Do the TNT at 37, not 30 (as the protocol used to sugggest)
2) Play with the Mg2+ concentration: easier (and a lot cheaper) to do
this if you
buy ordinary retic from Promega and make your own "home-made" TNT kit by
NTPs, T7 (GIBCO enzyme works well) and Mg according to the protocol of
Craig, NAR 20, 4987 (1992). Optimal incorporation of 35S-met doesn't
with optimal translation quality, and it does seem to vary according to
3) Transcribe capped RNA separately and titrate into plain retic: too
RNA often leads to increased amounts of internal initiation/premature
I've never tired this, but people tell me that titrating KCl can also
Hope this is useful.
Division of Virology
Department of Pathology
University of Cambridge
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