Something I recently learned from a friend: try changing the 4th base in the coding sequence to G (e.g. ATGGxx...) and the ribosome will like it better. We tried it recently on a frustrating construct and it worked like a charm.
________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Fernandez, Elias J [efern...@utk.edu] Sent: Friday, September 13, 2013 7:03 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] over-expression strategies Yes, the gene's been codon-optimized. The predicted mRNA structure appears stronger in the optimized version than the native, where it is high too. And, expression levels are unchanged. Elias ________________________________ From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Elias Fernandez [efern...@utk.edu] Sent: Thursday, September 12, 2013 10:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] over-expression strategies Dear CCP4ers, We’ve been struggling with little (nearly none) expression of our protein, in both E coli and with in vitro transcription/translation methods. It appears that our mRNA has high 2’ structure with a low dG (theoretically ~760kcal/mol). If this is indeed the source of our problem, are there any potential strategies to either disrupt the mRNA structure chemically (in E coli or in vitro) or with thermophile expression systems for expression at higher temperatures? Regards, Elias
