Overexpression

Bernard Heymann bheymann at bragg.bio.purdue.edu
Tue Feb 22 21:32:50 EST 1994


In article <2k9vmk$4ke at mserv1.dl.ac.uk>, Jochen Kruip
<KRUIP%DMSWWU1A.earn at earn-relay.ac.uk> wrote:

> Hi netters,
>  
> we have cloned a gene for a membrane protein involved in cyanobacterial
> photosynthetic electron transport. Now we want to overexpress this protein
> in E. coli to get larger amounts for a detailed biophysical examination.
> Is anyone out there who has experience with overexpression of membrane
> proteins? What about the different kits avaiable on the market? What are
> the advantages or disadvantages of a particular system? A special problem
> seems to be that expression of our gene is lethal to the E. coli host so
> we need a system based on a promotor under strong control. I don' t know
> if these questions are covered by a FAQ. Anyway all suggestions are
> welcome. I will post a summary to the net.
>  
> Thanx
>  
> Jochen Kruip, Institute of Botany, Schlossgarten 3, D-48149 Muenster
> Tel.: 0049-251-833803
>  
> E-Mail : KRUIP at dmswwu1a.uni-muenster.de

From: g3421101 at MUCC.MAHIDOL.AC.TH (Enrique Jose Labadan Frio):

>	You can try the T7 RNA polymerase/promoter system by S.Tabor (1990)
>in Current Protocols in Molecular Biology (pp 16.2.1 - 16.2.11).

We have cloned the colicin E1 immunity protein, a 113 integral membrane
protein, into the pT7-7 vector (which you can obtain from USB).
Overexpression of this protein induced with IPTG in BL21(DE3) seems to have
a ceiling at about 1.2 mg per liter of culture, with between 3 and 4 g wet
cell weight produced per liter (2YT medium). A similar ceiling have been
observed with another vector, and seems to be associated with some
intrinsic limitation. Apparently, the protein is not very toxic to the
cells and we have speculated that there is physically just not enough room
in the cell membrane to package so much protein. Wo have not seen any
inclusion bodies formed and it is presumed that any excess protein produced
and not efficiently integrated into the membrane is degraded by proteases.
It thus seems that pursuit of further increases in expression would be
futile.

Which protein have you cloned and how do you intend to purify it? Although
the purification of the immunity protein has been published, I am going
through some strategies now to be able to handle larger scale purification.
It may be useful to compare notes.


-- 
Bernard Heymann
bheymann at bragg.bio.purdue.edu



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