Good morning, I am trying to produce a recombinant protein. Unfortunately I one of the two restriction enzymes that I use do not cut at the end of the MCS. After the digestion 15 nucleotides of the MCS are expected to remain in the plasmid sequence. Of course they will remain also after clonation. Will these nucleotides that follow my candidate gene severely affect the quality of my recombinant protein?
If they are after the stop codon of your protein reading frame then it should have no effect.
Michael J. Benedik I do not have a stop codon in my protein reading frame, as I am cloning a part of a polyprotein. I will have a stop codon in a region of my plasmid after the his-tag. So, as these "extra" nucleotides are before the stop codon, they could affect the structure or the antigenicity of my protein?
In most cases I would not expect it to have any significant effect on your protein, especially if you are already changing the c-terminus by adding a his-tag. Just be sure that the number of added bases doesn't frameshift the protein so that the stop codon is still in frame.
Michael J. Benedik I will check it! Thank you very much for your help
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