Expression of a 300kDa protein possible?

Paul 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
> protein.

You don't say what you did get with the IVT: a smear of shorter products
or
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
kDa mark,
(albeit with quite a lot of shorter crap too), so 300 should be
possible.

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
adding
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
equate
with optimal translation quality, and it does seem to vary according to
the template.

3) Transcribe capped RNA separately and titrate into plain retic: too
much
RNA often leads to increased amounts of internal initiation/premature
termination.

I've never tired this, but people tell me that titrating KCl can also
help translation
quality.

Hope this is useful.

Paul Digard

Division of Virology
Department of Pathology
University of Cambridge






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