I want to overexpress a gene. For that I am going to make full construct of cDNA and insert it into a vector. I am just wondering whether it is necessary to include Kozak sequence before my cDNA. Does it cause any problems if I do not include it?
Yes it is advisable to include them in (eukaryotic expression system) cDNA. The Kozak consensus sequence plays a major role in the initiation of the translation process.You may get poor amount of protein if the sequence is not included.
It can't hurt to include one, so go ahead and do it.
If cost is not an issue, I would advise you to try making the insert with and without, and compare the expression level you get.
From my own experience in yeast, the Kozak sequence was not essential to get good expression for the inserts I wanted.
If it's from cDNA, then your gene will already have an endogenous ribosome binding site, which may or may not resemble Kozak's consensus sequence. In general I would look at how your protein is naturally expressed.
If it's naturally highly expressed in my cell type, like for example IG in lymphocytes, or albumin in hepatocytes, then I don't normally change it.
But if its naturally expression is low, or it's tightly regulated, or from a foreign species, I generally change it to match Kozak's best consensus sequence.
Which organism are you going to overexpress this cDNA in? Yeast? Plant? Mammalian system? If mammalian system, probably is good to include a Kozak sequence.
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