Overexpression of Eukaryotic proteins in E. coli

Alan Gerstein 76467.702 at CompuServe.COM
Tue Nov 3 12:15:00 EST 1992


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From: mcp at canctr.mc.duke.edu
Subject: Overexpression of Eukaryotic proteins in E. coli
Date: 2 Nov 92 16:38:10 GMT
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Dear Colleagues,

        I am currently trying to establish a system for overexpressing my 
favorite gene in E. coli.  I have a plasmid which contains MFG as a fusion 
with
the maltose binding protein (MBP - using the pMal-p2 vector from NEBL) which
is under the control of the Lac promoter.  I get expression at first
after transforming the plasmid into the host, but as I subculture the
little bugs, they decide to quit making my protein.  Now, I am
not streaking these guys on media with inducer, so they shouldn't
be unhappy about a foreign protein.  Also, upon induction, the cells
continue to grow even if they are making MFP, so the product isn't
lethal.  I have tried to express MFG in five different E. coli
lines: HB101, DH5a, BL21 (Novagen), HMS174 (Novagen) and Nova Blue.
No line was better at producing MFP.  Also, compared to the vector
with no insert, ie. just the MBP, the expression of my favorite
fusion is pitiful. I have tried a billion experiments with various
media and various growth conditions - temperature, amount of
inducer added, etc. and nothing seems to help. Plus, while I am
doing these experiments, the colonies that are on the plates in
the 'fridge are conspiring against me to refuse to express the
protein.  Does anyone have an idea as to:

1. Why should the amount of expression decline in cells grown
without induction. (The vector has a copy of the lacI gene on
it as well, so that leaky expression shouldn't be a problem -
and I can't see any expression in uninduced cells on a PAGE)

------------------

Have you considered the possibility that the protein is precipitating with 
time, perhaps in the form of inclusion granules?  You might want to check out 
Biotechnology 7: 1141 (1989) and Biotechnology 9: 725 (1991).

Good luck,

Alan



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