fusion protein - proteine-CFP (Mar/23/2006 )

hi I just create a fusion protein using clontech CFPN1.

I did a western blot and I see my fusion protein band and a strong band at the size of the GFP alone.
I did clone in the MCS upstream of the CFP.
so I was wondering why I got this strong band.

is it possible that the translation start not only on my inserted protein but also from the atg of the CFP.
in the CFP vector there is a kozak sequence.
I didn't put a kozak sequence in my insert.

since there a MCS in the CFPN1 I though by cloning in frame my protein without the atg with CFP will be ok.

any advice

thanks

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Could be, but there is possibly quite some space between the promotor and the Kozak sequence.
I don't know how this is regulated in the organism you use, but I would search it in either degradation of your protein (the CFP is mostly stable) or a protein "cut" site (for a specific protease for instance).

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hi
thanks
actually Iam using cos7 cells.

concerning the distance between kozak and promoter there is around 500nts because the CMV promoter is front of my insert then kozak then CFP.

looks like that: CMV-Protein-kozak-CFP

thanks

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