Expression in E.coli with rare codons
andy.phillips at afrc.ac.uk
Thu Mar 10 03:52:52 EST 1994
We're trying to express a number of Arabidopsis cDNAs in E.coli
to get large amounts of protein for biochemistry, NMR etc. We've
so far tried the pTrcHis vectors from Invitrogen, pUC19 and
pET-3d, but with no great success. We can detect active enzyme, but
get no impressive bands on SDS gels.
When we compare the codon usage of these genes, we find that they
use some codons for Ile, Pro, Arg etc which are very rare in E.coli,
particularly in highly-expressed genes. We've tried adding a plasmid which
carries rare Arg codons, with no effect.
How worried should we be about this codon usage? Is E.coli should worth
pursuing, by playing about with growth and induction conditions, or
new vectors? Or should we switch systems, and try a different one?
Any comments would be very welcome.
Andy Phillips (andy.phillips at afrc.ac.uk)
Long Ashton Research Station, Bristol, UK
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