Hello Sutapa, With codon optimisation you would get more and faster expression and likely even more insolubility in E. coli (less time for folding). To get the protein more soluble perhaps slower production might help. I guess you could try codon de-optimisation :-). Cheaper possibilities are growth at lower temperature, using a slower-growing E coli strain, using less IPTG or even no IPTG and just relying on the leaky expression, but perhaps you have tried all these already. Or another expression plasmid with tighter control or a different system like yeast. Codon optimisation for insect cells might be worth it if your analysis shows that the current sequence is really badly optimised. Or perhaps your protein needs a specific natural chaperone...it might be worth studying the natural production of your protein more before designing an expression strategy. Without knowing more details about your protein it's hard to give more help - you could try to contact other researchers studying similar proteins.
Greetings, Mark J van Raaij Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC calle Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://wwwuser <http://www.cnb.csic.es/~mjvanraaij>.cnb.csic.es/~mjvanraaij <http://www.cnb.csic.es/~mjvanraaij> > On 03 Apr 2017, at 07:49, Sutapa Chakrabarti <chakr...@zedat.fu-berlin.de> > wrote: > > 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 > > > > > >
