Hi Sutapa,
It is very difficult to predict protein solubility in advance. I have
worked with different proteins using 'native' sequences and codon
optimised 'synthetic' sequences. One of my protein was bHLH
transcription factor from Sorghum and codon optimisation didn't rescue
the problem of insolubility. I also tried MBP fusion for Arabidopsis
protein in which case the protein was soluble after centrifugation but
always end up in the void during SEC. My favourite method is
coexpression with interacting partner that also requires patience and
thorough optimisation but worth it. I have put the following reference
for further reading
'Codon influence on protein expression in E. coli correlates with mRNA levels'
Best of lucks
Sinta
Quoting Sutapa Chakrabarti <chakr...@zedat.fu-berlin.de>:
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
