bacterial expression vector

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Nov 30 10:13:33 EST 2000


>Hi
>
>We need to express a eukaryotic (plant) gene in bacteria with the aim to
>produce antibodies to the protein. Initial attempts (using the Novagen pET
>14b vector) were unsuccessful. We expect that our protein might be toxic.

In what way were your attempts unsuccessful?  That might be helpful to
know.  Given that you are planning on using the protein for Ab, then really
you have lots of possible options, I think.

If you are actually getting low level expression, then if you make a His-
or other tagged version, you could probably purify sufficient amounts for
Ab by affinity purification.  [Also, a tag is handy in a case like yours,
where you don't have Ab against the protein.  You can buy Ab against the
tag to help see whether you have low level expression.]

Have you checked whether your protein is insoluble.  I recently expressed a
plant protein via pET.  The expression level was pretty low-- hard to see
induction in total cell extracts, but quite obvious when I made
soluble/insoluble fractions.  In the end I purified protein from the
insoluble fraction from SDS-PAGE gel fragments, and was able to easily get
over a mg for Ab production.

Finally, since you are interested in Ab, you could try just expressing a
portion of the protein.  If the protein really is toxic, this would solve
that problem.  Also, in my experience, sometimes a portion of the protein
is easily expressed to high levels even if the entire protein isn't.  I've
even known people to express both halves of a protein seperately to raise
antibodies.

Hope these suggestions help.

Mike

Michael L. Sullivan, Ph.D

U.S. Dairy Forage Research Center
1925 Linden Drive West
Madison WI, 53706

(608) 264-5144 Phone
(608) 264-5147 Fax


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