Hello all,
I have constructed a plasmid which expresses my protein of interest in S2 cells. I have tagged the protein on its N-terminal with FLAG and on C terminal with V5 epitope. The protein expression was confirmed with anti FLAG antibody but I could not find any signal with anti-V5 antibody. After sequencing, I found that there is two base pair deletion which corresponds to C terminal of the fused protein that is supposed to express the V5 epitope. Now, my question is since there is no alternation in the coding sequence of my protein of interest, and it produces a band according to its molecular weight when stained with anti-FLAG, the activity shown by this protein in S2 cell (e.g. seen with luciferase assay) can be taken as the function of the protein itself or this can happen due to the premature stop codon of the V5 tagged portion of the protein. I want to skip the whole plasmid construction process if possible. Your answer will surely help.
It depents a little bit on the protein, most of the time, the terminal ends are not locted within the active centre and are not that importat for protein folding. In some instances however this IS the case. If you see the expected activity (which you do if I understood you correctly) the protein as such schould be fine, so the main consequence is, that you cannot detct your C-terminal V5. If this is not that important in any future step, than you are basically good to go.
However if you should want to publish the data it would be better, to work with a correct protein from the start instead of acceptig this deletion. If you however loose only a few amino acids, you can also think about just mentioning this fact in your discussion, since ther is a slight chance, it slightly changes the activity or has other effects.
If you have a "two-base" deletion I would recommend to start from scratch, mainly because you are changing the ORF (open reading frame). Consider also publication of your results: what the revieweer would recommend if he/she knows this piece of data concerning the protein used to obtain results in the submitted manuscript.
Thanks everyone for your valuable responses. I will go for the the whole construction process again to be in the safe side.
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