pET expression problems

Vladimir Svetlov svetlov at oncology.wisc.edu
Thu Sep 3 18:27:03 EST 1998


In article <35EE8C6D.93952BAD at plantphys.umu.se>, Adrian Clarke
<adrian.clarke at plantphys.umu.se> wrote:

Well, you already made a step in the right direction by using a tightly
regulated expression system for toxic proteins. Your protein size
differences can be explained by the instrumental error - SDS-PAGE gives you
a wrong estimate - or the product is indeed fragmented. You can check the
size by gel-filtration chromatography or mass-spec to make sure that the
size estimate is accurate. Then the possibility remains that your proteins
are truncated during synthesis because of suboptimal codon usage (clusters
of rare codons can cause premature termination of transcription) or
unstability of the mRNA (that all gets chewed up to the same tertiary
structure somewhere). If the rare codons are the problem you'll have either
to bacterialize the codon frequency according to E. coli preferences or use
cell-free system of transcription/translation. First is tedious second is
not very preparative.

Regards,
V.



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