thanks to all for the replies. the protein of interest is a membrane associated (lipoprotein) but i express this without the signal sequence in the cytoplasmic context. also it is not his-tagged, to address the debate of protein crystallize with or without the tag i presume as one reason. also the protein is stable without the tag. the purification was lengthy having amm.sulfate ppt, ion exchange and size exclusion. also the MW (and pI) was very similar which prolonged this identification but when the fractions are yellow in color, that arouse curiosity. ESI-MS had two charge state envelopes. on one note i am happy the cell compensated then it enabled overexpression of desired protein to a greater extent that may have be toxic to cell otherwise.--Karthik
On Tue, Dec 15, 2009 at 2:11 PM, James Holton <[email protected]> wrote: > 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. >> > >
