Dear Sutapa,
As already pointed out, codon optimization for increasing the solubility
is not really a viable option but it can be for production /per se/ in
insect cells.
Among others, slowing down growth, changing the construct or
coexpression (of for example foldase/chaperone) or witching to a
homologe can be viable options though.
PLease note that instead of slowing down the growth of /E. coli /by, for
example, lowering the temperature, add the IPTG at a very late stage of
the growth, about 1.5 hours before reaching the stationary phase (late
log phase) when cell density is really high. Big advantage is, lots of
cells that produce the protein of interest slowly (because of
methylation processes of ribosomes and elongation factors etc. if I
recall correctly) and harvest after about 1 hour.
- J. -
Dear all,
We’re trying to express and purify a 1000 residue long protein and
have run into the problem that it is completely insoluble when
expressed in E.coli and is not expressed at all in insect cells. The
usual tricks for improving solubility in E.coli, such as addition of
GST/MBP tags, optimising expression media and induction conditions
and use of different cell strains, have not led to any improvement.
We are now looking into ordering a codon-optimised synthetic gene for
this protein and are trying to decide whether it would be worthwhile
to codon-optimise for expression in E.coli (given that the protein
was expressed but not soluble) or if we should attempt baculovirus
expression again with a gene that has been codon-optimised for insect
cells.
My question is:
has anyone observed an improvement in the solubility of their target
protein using a codon optimised gene?
I know of several instances where the use of a codon-optimised gene
has led to expression where the native gene sequence did not but am
unable to find any references for improvement in solubility. Since
codon optimisation significantly alters the translation rate of a
gene, I believe this should affect solubility as well; but I’d like
to know what the community thinks/has observed before I order an
exorbitantly priced gene!
Thank you in advance,
Sutapa
--
Sutapa Chakrabarti, Ph.D.
Institute of Chemistry and Biochemistry
Freie Universität Berlin
Takustr. 6
14195 Berlin
Germany
Phone: +49-(0)30-83875094
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Dr.math. et dis. nat. Jeroen R. Mesters
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(Shakespeare's Macbeth, Act I, Scene 3)
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It is invariably the case that high resolution X-ray structures show
significantly better agreement with solution observables such as
coupling constants, 13C chemical shifts, and proton chemical shifts,
than the corresponding NMR structures, including the very best ones.
Hence, in most cases, a high-resolution crystal structure (< 2.0 Å)will
provide a better description of the structure in solution than the
corresponding NMR structure (Kuszewski, Gronenborn & Clore, 1996,
Protein Science 5:1067-80)
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