Dear Puspita

i do not undertand your question.

Is the protein degraded at the N-term when is it expressed with out C-term TAG, while the TAG seems to protect it with the degradation? Or if is degraded at the N_term the fusion proteins?

best regards

Manuele

Hi Puspita,

You should check your insert construct to see if there is a section in the beginning that would cause a frame-shift when tagged on the N-terminal side. This would lead to a nonsense protein production and thus degradation. As well, is the insert "Closed" (has a stop codon) or "open" (no stop codon). If you have an open construct, the C-terminal tagging will work (as will the N-terminal), but when tagged at the N-terminal, there will be some extended regions that are translated beyond the insert sequence. This region may trigger protein degradation as well.

Reuben

Dear Manuele,

Thank you for responding to my query. I would like to tell you that the protein contains an HA tag at the C-terminus, we are getting a truncated version of the protein on immunoblotting using HA-tag, hence, we are assuming that it is a degradation product that reflects only the C-terminus, while the N-terminus is getting degraded.

I would like to understand if our assumption is correct, then how is it possible that only the N-terminus is getting degraded while the C-terminus remains intact with the HA-tag and also why not the full-length protein is getting degraded is my another question.

You have added an interesting statement in your answer that the fusion tag is protecting it from degradation, can you please elaborate on this?

Please share your take on this.

Thanks

Puspita

Hello Reuben,

Thank you for adding an answer. I will surely check the construct based on your suggestions.

Thanks

Puspita