4 replies to this topic

[Thidarat Au]

Hi everyone,

I'm new in molecular cloning and protein expression. pET20 is available in my lab so I expect to use it. It produce C-ter-His-tagged protein. I plan to insert at NdeI-EcoRI site. and I notice that almost twenty amino acids (include His tag) are fused with my protein. I don't need to remove His tag but I wonder that whether these amino acids affect my protein property. (I need to determine only phenotye of the clone harboring my gene)

Thank you everyone,
sis029

[phage434]

Short answer: there is no way to know.  You might get some insight from looking at a 3D structure of your protein, or even from checking the helix/turn/beta 2D propensity, but you have to try it.

[Thidarat Au]

thank you. your answer is useful for me. I think I'll try 2D first.

[bob1]

It certainly can affect your protein. You should also tag the protein on the other terminus to check changes in distribution.  If you have antibodies against the native protein, compare the tagged with untagged.

[Thidarat Au]

I dont have antibody. but I think of tag on N-ter is easier for me, thank you:)