 |
 |
There is also lack of information here. Do you really expect this protein in soluble fraction? Is it a membrane associated or transmembrane protein? Well, I don't have any experience in protein expression using codon optimization. However, Considering the fact that in E.coli it is getting expressed in insoluble fraction, I would recommend to co-express this protein with groEL/ES system. I have plasmid expressing groEL/ES operon. On Mon, Apr 3, 2017 at 12:22 PM, Hansman, Grant < [log in to unmask]> wrote: > Hi, > > Why don't you try adding GST/MBP tags first? This is a easy quick test. > > We have a nice fusion (MBP) vector for e.coli expression if you want. > > Grant > > From: Sutapa Chakrabarti <[log in to unmask]<mailto: > [log in to unmask]>> > Reply-To: Sutapa Chakrabarti <[log in to unmask]<mailto: > [log in to unmask]>> > Date: Monday 3 April 2017 08:49 > To: "[log in to unmask]<mailto:[log in to unmask]>" < > [log in to unmask]<mailto:[log in to unmask]>> > Subject: [ccp4bb] Using a codon-optimised gene to improve protein > solubility > > 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 > -- Vipul Panchal Senior Research Fellow, Respiratory disease and biology, CSIR-IGIB (M)-9540113372