I saw something like this once that turned out to be CAT
(chloramphenicol acetyltransferase) denatured and entangled with a
massive amount of RNA (which was yellow). I was not using
chloramphenicol, but an examination of the vector lineage reminded me
that the gene was in there. This aggregate was very soluble and also
thermally stable (falling apart around 80C), which had me going for a
while because I was after a thermophillic protein (which, BTW, never did
express but would have been very close in MW to CAT). I imagine other
proteins could fall into to the overproduction of mRNA that is inherent
to "greedy" expression vectors. Sounds like your story may be
different, but I agree that overexpression can indeed do weird things to
the guts of E. coli.
-James Holton
MAD Scientist
Karthik S wrote:
Hi, I apologize immediately that this question is not directly related
to crystallography but the protein i am trying to overexpress is
eventually for that purpose. i understand the huge knowledge-base of
people here experienced in protein expression/purification and would
appreciate any insight in the following.
i have the protein to be expressed on a pETBlue2 vector transformed
into Origami (DE3) pLacI cells (the protein has one disulphide bond).
Upon purification there is another protein that is also overexpressed
to the same level as my protein of interest. Is this normal for
another unintended protein to be overexpressed along with the one on
the plasmid? (It is not a breakdown product). I did manage to separate
this second protein that turned the fractions yellow. A UV spec
indicated double peaks between 300-350nm indicating the presence of
oxidized flavin (that would explain the yellow color). A ESI-MS to
identify the unknown protein by its MW gave Alkyl hydroperoxide
reductase subunit F (MW=56177) as the closest possible match. now is
this to maintain a redox balance inside the cells? i understand the
Origami cell line to be deficient in thioredoxin reductase (trx) and
glutathione reductase (gr).
----
Thanks, Karthik
Graduate Student, Biophysics
U-M, Ann Arbor.
