Is the Kozak sequence really needed for cloning to express the gene? - (Jul/24/2012 )

Hi Friends,


I am cloning my gene of interest in pAAV-DIO vector to cell specific expression in brain. However, my gene doesn't present any kozak consensus sequence upstream and downstream of the ATG naturally. The Kozak consensus sequence plays a major role in the initiation of the translation process. Therefore, I can add kozak sequence upstream of ATG; however it is not possible in downstream. This manipulation might work. Even without the Kozak sequence, the translation efficiency of the protein might drop, or will drop. I do not have experience with the mammalian expression system. Is the Kozak sequence really needed for cloning?

Kindly suggest me.

Thanks,
Ramesh

Usually no, but it often helps.

To add to bob1's comment, I contacted Promega last year about the same issue. They said their pCIneo already has CMV promoter, so having a Kozak's sequence is really not needed. But I personally think that having the GCCGCCACC before the start codon will improve expression. The problem is if you can put this sequence in your primer, because your primer will become longer, and it will affect your Tm. I normally just add ACC, because most of my genes start with ATGG. My colleagues say it's already sufficient.

Thanks you very much.

I added GCCACC before ATG in the primer. I hope this should work.'

Thanks Again.