I need to flag-tag my protein at N-term, and while designing the primers i was confused whether to remove the start codon of the protein, falling immediately after the flag sequence ends and the protein sequence starts.
Dear Shradha Surin
Yes, you can remove the starting met of your GOI since the starting Met will be present in the N term of the tagging protein.
I suggest you to add between the TAG amd the protein a protease cleavage site as TEV , Fatt. Xa
best regards
Manuele
Manuele Martinelli thanks for your suggestion. Even i was having similar thoughts, but i was wondering whether the absence of Met at the beginning of the protein wouldn't affect its folding or functionality.
Dear Shradha Surin
each proteins has it is own histoy but to my knowledge in most cases the n terminal methionine is cleaved in vivo and do not have a role in protein function
https://pmc.ncbi.nlm.nih.gov/articles/PMC5663234/
best
Manuele
Manuele Martinelli sorry for late reply. Thank you , i had no knowledge about it
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