I intend to fuse my gene of interest (apoptin) with GFP using pCAMBIA1302 vector. I figured that I can do that by adding NcoI and SpeI restriction sites to 5' and 3' end of my GOI using PCR and later insert it into the plasmid.
However, as you can see in the image, 3 base pairs within SpeI restriction site do not overlap with the GFP gene and thus will be translated as an amino acid that is neither GFP's nor the GOI's. Will it disrupt my fusion protein structure/function? Thank you.
I think you should be fine with this. Many times it is wise to put a linker region between your desired protein and the desired tag (here gfp). This helps to ensure proper folding and hopefully functionality between the protein of interest and the tag. Your linker amino acid here will not produce a stop codon, so the gfp should be transcribed, but you should always check to ensure your desired protein remains functional when adding a tag on the end of a protein. Just ensure that you do not include the stop codon in the PCR primers for your protein of interest, otherwise the gfp will obviously not be transcribed.
Rafiif Wasis Ibaadurrahmaan As far as the open reading frame of the fusion proteins are not disturbed, the function would not be affected.
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