[Arabidopsis] At gene expression in E.coli
(by jiangyqcn from hotmail.com)
Thu Oct 9 23:42:28 EST 2008
I ever tried to express a few At transcription factor (TF) genes in E.coli using the Novagen pET32a(+) vector and BL21(DE3) as well as Rosetta-gami strain. Only one of them (aa shorter than the others) was successfully expressed in the Rosetta-gami strain, suggesting that codon bias is possibly a problem for plant TFs exp in E.coli. However, the sole expressed TF was not in the soluble fraction; it is in the form of inclusion body, although I induced it at 30C for only 3 h. It takes a lot effort to get it out from the inclusion body and then refold it. So far, I did not see much use of using E.coli to express a TF protein. Another lab mate also got an unknown protein gene expressed in the inclusion body. A friend saw a fungal gene expressed in the inclusion body too. I ever read a paper describing a strategy in E.coli(not too sure) that adds a tag to target protein and secrete the protein into the medium, is any company selling this type of vector? What made I very doubtful is I saw a published paper expressing one of the TF genes I failed to get exp in E.coli and they succeeded with BL21(DE3) strain. I checked their "Materials and methods" section and found that the IPTG concentration they used was also 1 mM, the same as I ever used. Other than this, no more detail was included in their paper; they just saw "according to the manufacturer's manual". No idea whether that TF was expressed in soluble form or in inclusion body. No info was included in the supplemental data. Were they cheating me?
How about in yeast? What are the working yeast vector and strain (vendor?) for expressing At genes? Does anyone ever get an At gene expressed in the soluble form in yeast? How does yeast exp system compared to E.coli system?
Thanks for your reply.
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