As you know, when we express heterologous proteins from different organisms in E. coli, we might encounter some proteins utilize many rare codons in E. coli so the expressions are extremely low. I struggled a long time with this kind of expression until I found out this fact (and amazingly that no one reported it before). When I used Rosetta(DE3) strain I got very strong expressions. Knowing that the Rosetta(DE3) strain uses the pRARE plasmid which overexpresses several rare tRNAs for the codon optimization in E. coli, that's very helpful if I combine this area of pRARE into my construction. However, I can't find the exact sequence anywhere, seems like the company want to keep it. I decided to isolate and sequence pRARE, start from the cat and p15A sequences. I didn't finish sequencing the whole plasmid now, but surprisingly that I didn't see any sequence of tRNA as they mentioned in the map. Actually downstream the p15A ori are a fragment from T7 phage containing lysozyme and protein 3.8 sequences and then tetR sequence. I'm starting to be confused so I want to find out more information from someone else. And here is the map that Novagen provided:
Thank you very much in advance!!
Edited by Quasimondo, 06 March 2011 - 06:37 PM.