My Questions are:
1. Should the gene of interest be directly after the IL-2 sequence and if that is the case, should it have its own start codon (ATG)?
2, if it has to have its own ATG, would that prove to be a problem during protein expression? Is it possible that ribosome might skip the start codon of signal sequence and start at the gene of interest. Would adding a Kozak sequence in front of IL-2 prevent that?
Hi Prerna,
1. the gene of interest should be placed immediately after the signal sequence for the production with a native end. You don't need the ATG but can have it if you want. Any unwanted amino acids between the end of the signal and the start of the protein will remain, which may be a problem for your studies.
This raises some question. Is your gene of interest normally secreted? If so then does it have a signal sequence? The best thing would be to replace that signal exactly with the IL-2's. If it's normally not secreted, then why are you wanting to secrete it?
2. internal ATGs don't usually cause a problem, most genes will have several (both in and out of frame). However it is important that the start ATG has a good Kozak sequence, otherwise there will be a higher chance of false starts.
-Richard
Hi Richard
Thanks so much for your insight. Actually I will be injecting my construct eventually in mice and for that it needs to be secreted to get an immune response. And my gene of interest doesn't have its own signal peptide, so we are using IL-2 signal peptide.
-Prerna
Hi Prerna,
Ok, I understand. Watch out for Cysteines that could form bonds when secreted, that normally wouldn't.
Good luck.
-Richard
- Badges
- Science method