My protein is cloned in pEt 41a vector. I have shifted it from pEt 21a to pEt 41a by designing the primers with Kpn site. So that we may remove the tag later on using thrombin. But in this way there are 25 extra nucleotides before Aug of the protein. Will these Extra nucleotides affect the structure of the protein after purification?
Your description of the problem is a little unclear. Are the extra nucleotides actually in the open reading frame (such as between the tag and the protein) or are they upstream of the protein start and not translated at all.
If it is the first of these, then extra amino acids on one end or the other end of a protein normally would not affect structure. There are some rare instances where it might, but not usually. However 25 nucleotides is not a multiple of 3, so if that is an exact number then you might have shifted the reading frame.
On the other hand, if the 25 extra nucleotides are just upstream of the AUG start codon but not being translated, then they would have no effect on the protein at all. However they might be altering the distance between the ribosome binding site so would impact translation efficiency.
Adding 25 nucleotides may cause frameshift, you shoul add or remove order of 3x amount of nucleotides. Also this frameshift even seems like not effects the protein , promoter region or Shine Dalgarno sequences might be effected. This might effect the expression level. Could you share your results if you did this?
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