Proteins in E. coli coexpressed with recombinant proteins?

Achim Recktenwald, PhD ARecktenwald at StressGen.com
Thu Jan 25 19:39:12 EST 2001

A couple of years ago at a different job, we sequenced a co-expressing
protein that ran also ~34kDa; it turned out to be the initiation factor Tu.



"Emir" <ekhatipo at NOSPAMmidway.uchicago.edu> wrote in message
news:RH_a6.384$x3.4740 at uchinews...
> Hi,
> I was wondering if anything is known about the indigenous E.coli (or other
> bacterial) proteins that are expressed in response to expression of
> (poisonous) recombinant proteins.
> Here's my case. I am expressing a 6his-tagged yeast DNA-binding protein in
> E. coli (BLR, a RecA- strain) from a T7 promoter-driven expression vector
> (pET-11). The protein is a homologue of RecA and that is why expression in
> RecA- E.coli is important to prevent crosscontamination of the target
> protein. In addition, overexpression of the recombinant protein is highly
> toxic for E.coli, that is why I am using lambda CE6 phage to induce
> expression in the non-DE3 strain (strain with pLysS shows same result in
> terms of what described below).
> On Westerns with primary antibodies against the recombinant protein, I
> always see 2 bands, one of the target protein (~37kDa) and another of some
> unknown protein (~34kDa) that happens to bind same antibodies. That 2nd
> protein is present both in non-induced, and induced cells, as well as it
> present in the non-transformed cells (i.e., the ones not bearing the
> expression vector, ! ). Thus, the protein #2 is indigenous E.coli protein.
> Interestingly, the level of that protein increases upon induction of
> expression of the recombinant protein, almost to the same level as the
> recombinant protein, as seen on SDS-PAGE and on Westerns. Since the
> #2 is present in non-transformed cells, its appearance in transformants
> coinduction with the target protein cannot be explained by proteolysis of
> the latter or secondary translation initiation site on the plasmid
> construct. RecA- strains still have E. coli RecA homologues DnaB and RadA,
> but both proteins have MW higher then 40kDa (vs. 34kDa of the protein #2)
> I am trying to understand what is the nature of that protein #2 and is it
> possible that it is a certain defense agent synthesized by the host cells
> neutralize the poisonous recombinant protein? It could be that this
> neutralizing agent binds the recombinant protein and inactivates somehow,
> i.e., some sort of bacterial antibody (SOS response, etc.). The binding
> hypothesis could explain the crossreactivity of antibodies. I am sure that
> everybody noticed that almost in every case of recombinant expression, one
> sees a number of other protein bands that become more intensive on stained
> SDS-PAGE gels in induced cells vs. non-induced, whereas the majority of
> bands has the same intensity.
> Any comments, ideas, references would be much appreciated.
> - Emir

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