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).
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Thanks, Karthik
Graduate Student, Biophysics
U-M, Ann Arbor.
