Kozak sequence between GST and gene of my interest - There is a kozak sequence between GST and my gene. Does it hurt? (Jun/19/2007 )

Hi folks,

The purpose of the experiment is that I have to purify GST fused kinase protein and do some in vitro kinase assay.

So, I cut MCS site of GST vector and kinase gene insert, and try to ligate them. The problem is that;

If I ligate insert and the GST vector,

ATG---------ACCATGG-------

start codon start codon
of GST gene of insert

there is a kozak sequence inbetween (ACCATGG).

Do you think protein without GST will be expressed significantly a lot, compared to fused one and may affect something bad for the fused protein due to this kozak sequence? Also, there is no kozak sequence at the start codon of GST, so I wonder if competitively protein expression without GST may be dominant or not.

A lot of thanks to all of you.

I presume you are using a commercially available vector containing the GST? if so, I would not worry about lack of Kozak at the beginning of the GST or about the 'internal' Kozak sequence. I would expect that the vast majority (>99%) will be GST-kinase and not just the kinase. Best assay is of course to test the expression of your GST fusion...