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 [cid:image001.jpg@01D2AC7E.67A52A60] 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
