expression without kozak? - (Jul/25/2005 )

Hi there,

I want to try to make a recombinant HIV virus by cotransfection of a proviral clone deleted for my gene of interest and an expression vector with my gene of interest. In this way I hope to create virusses that are only capable of a single cycle infection (the proviral genome does not contain an essential gene so it shouldn't be able to finish a complete replication cycle).

My problem is that in the HIV genome (which is very diverse) my gene of interest does not have a kozak sequence near the start codon (it is transcribed as part of a multiple spliced RNA molecule), and due to the diversity I can not get it into my primer either.

So my question is if the kozak sequence is 100% necessary to have a high expression?

If it is, is it possible to make a longer primer so that I add a kozak 5' to the ATG of my gene? (I know kozak already contains an ATG so that would make two start codons in a very small distance, but the first part of my gene just is a signal peptide, so maybe it doens't mather too much to make that peptide a little longer as long as I remain in frame?)

Thanks!

Hi,
You'll find that some people will tell you that you don't need a Kozak sequence because they have used constructs without them and they work fine. I would disagree a little because while it may express fine without Kozak it may not. You won't know untill you try. If you don't add a Kozak its a crap shoot. you may get low medium or high expression depending on how well the ribosome likes the random sequence that happens to be around your ATG. Personally I never risk it. I build most of my constructs by engineerining restriction sites onto the ends of the primers so that you'r PCR product has the sites of interest (restriction sites or kozak etc).
The answer to your question: "is it possible to make a longer primer so that I add a kozak 5' to the ATG of my gene?" is yes except that you don't need to have two ATG's. You can engineer the primer however you want. The engineered part of the primer will not be complementary to your template so just start the comlementary part of the primer wherever you want. For example:


nnnngaattcgccgccaccatgxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx
XXXXXXXXXXXXXXXXATGXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX


The lower case letter represent your primer. The 4 n's are there because most restriction enzymes can't cut right at the end of a strand of DNA. gaattc is the EcoRI site. gccgccaccatg is the Kozac sequence.
The upper case letters represent your template.
Sometimes when I teach this to students I'm working with they worry that the engineered overhang will remain single stranded but if you think about the way PCR works you'll realize that it gets filled in and becomes double stranded.