Dear Sutapa There has been wonderful suggestions, particularly from Bert, size matters!
Considering that all is in order, i would share my experience with retroviral integrase, a sparingly soluble protein when expressed in E. coli. The protein exists in mammalian cells as a multi-protein complex assembly. When expressed alone, many of the surface-exposed residues were available for non-specific interaction. Surprisingly, mutation of one hydrophobic residue to lysin turned the table around, and we got ample protein in soluble form. We also tried super-charging algorithm from Rosetta, as well mutation of those surface-exposed residues that are involved in the protein-protein interaction. There must be a way to express these challenging proteins in E. coli. We always start with the easy and handy tricks, but some proteins are just notorious, and demand more attention! Best of luck! -Z Zaigham Mahmood Khan, PhD Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York On Mon, Apr 3, 2017 at 6:30 AM, Noam Adir <n...@ch.technion.ac.il> wrote: > Dear Supta, > > > > If I may make another type of suggestion, we have had success in > crystallizing sparingly soluble proteins in the presence of up to 2M urea > (Dines et al. Journal of Structural Biology, 2007). It is enough to avoid > non-specific associations, but not enough to denature the protein. It is > certainly worth a try. > > > > Noam > > > > > > *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of > *Sutapa > Chakrabarti > *Sent:* Monday, April 3, 2017 9:50 AM > *To:* CCP4BB@JISCMAIL.AC.UK > *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 <+49%2030%2083875094> > > > > > > > > > > >
