kozak sequence - (Jun/11/2008 )
Here is the link to the sequence I want to express my protein from. http://www.ncbi.nlm.nih.gov/entrez/viewer....mp;id=113037609
Can some one please tell me if I need to add Kozak sequence before ATG in this .
I dont have a G followed by ATG in my sequence does that means I need to add this G , wont it change the protein sequence?
Also when I will add the RE sites and Kozak in primers can that also change the protein sequence. DOes that mean I need to add extra bases to make sure that they are in the triplet and express some normal protein before start codon.
Please reply If anyone knows the answer. I need to start this work as soon as I can.
many thanks to you guys for helping me all this while.
You need a Kozak sequence before the ATG - we use GCCGCCACC for expression in mammalian cells and this works fine. You don't need a G after the stop codon. Like you said, if you add this you will change your protein sequence and you can't do that. The protein sequence starts at the ATG. Anything on the 5` side of the ATG will not be translated into protein. You don't need to worry about frame or "triplet" as you've called it. This is only when you are expressing tags (that are found in your vector) along with your protein sequence.
The structure of your forward primer should be:
XXXXXX - RE site - GCCGCCACC - ATG (start codon) - gene
X = flanking bases required for proper restriction enzyme cutting
One of the restriction site which is commonly used in designings expression vectors in NcoI CCATGG which is by itself a very good way of getting a restriction start and at the same time an ATG an a Kozak sequence, so you just modify the beginning of your protein on the second amino acid only
Agree with killerkoz17, my kozak is (gcc)gccRccATGG, where R is purine (A or G). The (gcc) can be omitted and the G after start codon can be ignored. with just gcc(g/a)ccatg, it already work. Don't worry about frame shift at the 5' region because translation start site will be at the ATG, right after the gcc(g/a)cc and your RE site is upstream from that. If you're N-term tagging your protein, don't add the kozak sequence. The kozak sequence must already have been included in the vector.
Thanks to all of you , I think I understood the concept . hope it works out for me.